Poster Abstracts Page

The poster session for the 2018 Regenerative Medicine Workshop will be held on Thursday, March 22 from 5-8:00 p.m. in the Sweetgrass Pavilion Conference Center. Poster boards, easels and pins will be provided. Poster board dimensions are 60in x 40in (posters may not exceed these measurements) and posters may be displayed vertically or horizontally. All posters can be set up at check-in on Wednesday, March 21, in the foyer area of the Sweetgrass Pavilion (see printed program for your assigned poster location) but no later than Thursday, March 22 at 10:30 a.m.

J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida (1); Department of Biomedical Engineering, University of Texas (2)

Spinal cord injury (SCI) is permanent and debilitating and currently, no treatments are available for the repair of damaged nerves. Breakdown of myelin sheath causes the release of inhibitory molecules such as Nogo-A, myelin-associated glycoprotein (MAG), and oligodendrocyte myelin glycoprotein (OMgp). These inhibitors bind to the nogo receptor present on the surface of damaged axons and prevent regeneration after injury. Regeneration of injured nerves is a slow process and may take few weeks to months, therefore sustained and localized release is desirable. Hydrogels are a commonly studied biomaterial platform to deliver therapeutics locally, however, due to high water content, they tend to be permeable, causing rapid release of encapsulated drug. This fast release may cause serious complications due to local overdose and also results in wasting of expensive drugs. Therefore, several labs have currently focused their efforts on controlling the degradation profiles and improve the sustained release of encapsulated drug. In this research, we designed a novel oligonucleotide hybridized hydrogel system using ssDNA oligonucleotides (immobile species) as antisense sequences to fine-tune the release of anti-NgR aptamer (mobile species) to enhance axonal regeneration after SCI. The anti-NgR aptamer is ssRNA with high affinity to bind to the Nogo receptor (NgR) present on the surface of axons. This aptamer competes with inhibitors for binding to NgR, thus promoting axon elongation in the inhibitory environment.

Oligonucleotide hybridized hydrogels were produced by covalent conjugation of anti-sense oligonucleotides molecules (ssDNA) on hyaluronic acid (HA) based hydrogels. Hybridization of oligo sequences was achieved by oxidation reaction causing the formation of the disulfide bond in thiolated-HA hydrogels and using EDC-NHS chemistry resulting in amide bond formation in methacrylated-HA hydrogel systems. Rheological measurements under shear stress were performed to match the mechanical properties of gels to those of native spinal cord tissue. Antisense oligonucleotide sequences with varying complementarity were successfully designed using NUPACK and Oligoanalyzer software packages and an increase in binding affinity was observed with increasing complementarity. The optimal concentration of aptamer required to promote neurite outgrowth in presence of inhibitors was determined in an in vitro experiment. In vitro release studies were performed to assess the ability to control aptamer release from the hydrogel based on binding affinity between the aptamer and antisense sequences. Currently, experiments are in progress to test the efficacy of aptamer released from hydrogels in promoting neurite outgrowth using dorsal root ganglion neurons. Future work will examine the efficacy of binding affinity based aptamer release in an in vivo environment using a rat SCI model.

Skeletal muscle has a remarkable regenerative capacity due to its maintenance of a robust pool of muscle stem cells. However, after a vast loss of skeletal muscle due to traumatic injury or surgery this regenerative response is lost, causing chronic functional deficits. The standard of care for these large muscle injuries, known as volumetric muscle loss (VML) injuries, have poor clinical outcomes, creating a need for tissue engineered regenerative therapies. Although it is generally accepted that there will not be functional regeneration after the loss of a significant portion of skeletal muscle, a critical size at which stem cell niche components are sufficiently damaged has not yet been established in muscle. In our study, we utilized multiple VML injury sizes of 2, 3, or 4 mm diameter biopsied tissue from the mouse quadriceps. We used 4 separate animals for each injury size and outcome measure and one-way ANOVA was used for statistical analysis followed with Tukey’s post-hoc testing for analysis of quantitative data (wet weight, fiber cross-sectional area, fibrosis area, neuromuscular innervation, vascular volume) where p<0.05 was considered significant. 2, 3, and 4 mm injuries resulted in 5, 15, and 30% reduction in the total quadriceps mass, respectively. At 14 and 28 days after injury, we examined general morphological differences and fibrotic response of the quadriceps using histological techniques, and revealed significant differences in the morphology and fibrotic area between each injury size. These cross-sections were also immunostained for the myofiber membrane protein dystrophin and myonuclei, and observed significant increase in the number of small, regenerating myofibers with centrally located nuclei with increasing injury size. These results collectively suggested that a 3 mm injury was at the critical threshold, as it resulted in significant increase in fibrosis but non-significant loss of mass from the contralateral control. Muscles with 3 mm injuries exhibited significantly decreased neuromuscular junction (NMJ) synaptic overlap and significantly more newly forming acetylcholine receptor clusters in and around the injury as compared to the control. Further assessment of angiogenesis via micro-CT analysis revealed a significant increase in vascular volume after VML injury as compared to the control. Taken together, these data indicate that spatial and temporal control of the fibrotic and neuromotor response are critical components in the injury space and could be potential therapeutic targets, as these seem to be the most dysregulated components of the muscle stem cell niche after VML injuries.

3. Seth Andrews

Stimulation of Exosome Producing Cells Alters Immune Modulation

Seth Andrews (1,2); Timothy Maughon (1,2); Steven Stice (1)

Regenerative Bioscience Center, University of Georgia, Athens, Georgia (1); College of Engineering, University of Georgia, Athens, Georgia (2)

Exosomes, small membrane bound vesicles secreted from nearly all cells, have recently attracted interest as delivery vehicles for therapeutics. Stem cell generated exosomes are of particular interest, as they have been shown to recapitulate some of the effects of the cells themselves. Stem cells are responsive to their environment, with mesenchymal stem cells (MSCs) changing to an “activated” phenotype in the injury microenvironment. This study examined the effects that acidity, hypoxia, and inflammation have on exosome production in neural (NSC) and mesenchymal stem cells, as well as the immune modulatory potency of those exosomes, compared to other cell secreted factors.

Human NSCs and MSCs were cultured in serum free media under inflammatory, acidic, hypoxic, or normal culture conditions. Conditioned media was collected for ultrafiltration to isolate exosomes. Both exosome depleted media and exosomes were frozen at -20C. Vesicle size distribution and concentration were measured via Nanosight. Human peripheral blood mononuclear cells (PBMCs) were obtained from healthy donors, stained with CFSE, and stimulated with anti CD3/CD28 Dynabeads for three days in the presence or absence of exosomes or conditioned media. The PBMCs were then harvested and stained for CD4, CD8, and CD25, or CD4, CD8, TNF-a, and IFN-y. Flow cytometry was used to obtain the mean percent positive and mean intensity for CFSE, CD25, TNF-a, and IFN-y in CD4+ and CD8+ cells as a measure of the immune modulation of T cells.

Preconditioning with different aspects of the wound microenvironment had dramatically varying effects on exosome production in two stem cell types. MSC exosome production was greatly increased by acidic or inflammatory culture (p<0.05, p<0.01 respectively), while NSCs had little response to the same environment. Hypoxia had little effect on the number of exosomes produced by either cell type. These findings underscore the importance of the cell microenvironment on exosome release, and the immunomodulatory potency of the exosomes will be investigated further by the previously mentioned flow cytometry experiments.

Annually, over 5.7 million Americans are diagnosed with intervertebral disc (IVD) pathologies including herniation (IVDH). IVDH detrimentally alters the native architecture, composition and mechanics of the IVD, and more specifically disrupts the annulus fibrosus (AF) region of the IVD. An ideal annular repair strategy must above all function mechanically to re-establish AF function, but should also support long-term tissue regeneration. To address this, our lab has developed an acellular multi-laminate collagen-based implant; an annulus fibrosus repair patch (AFRP), whose architecture and mechanical properties have been shown to mimic that of native human AF tissue, which demonstrates the ability to support human adipose derived stem cell (ADSC) viability. The objective of this research was to further evaluate the regenerative potential of our AF implant. This was assessed by seeding AFRP implants with human ADSCs and culturing the constructs for up to 56 days. ADSC viability was confirmed via Alamar Blue assay and the cells were assessed for their ability to differentiate into an AF cell-like phenotype on the implants via real-time reverse transcription PCR for AF markers (collagen type I, V, XII, decorin, prolyl-4-hyddroxylase). New collagen deposition was assessed via Herovici’s staining, and the resultant biaxial mechanical properties of the ADSC-seeded implants were evaluated. Herein, cell viability of AFRP seeded implants was confirmed via a statistical increase in cellular metabolism with increasing time in culture. Gene expression of collagen XII and decorin demonstrated an upregulation at days 3 and 15 compared to day 0 hADSC controls. ADSC seeded implants also demonstrated a statistical increase in collagen I expression between days 3 and 15. Herovici’s histological staining confirmed the presence of new collagen deposition within the AFRP implants by day 56. Moreover, a statistical increase in the circumferential biaxial mechanical properties of ADSC seeded implants was observed. In summary, we have demonstrated the ability of the AFRP implants to support ADSCs viability and differentiation towards and AF cell-like phenotype which resulted in the deposition of a collagenous matrix and biaxial mechanical properties which mimic native human AF tissue. Taken together, this data demonstrated that our AFRP implant can be used clinically to promote IVD repair and long-term regeneration which could lead to the utilization of a less aggressive palliative surgeries and the minimization of IVDH reoccurrence rates.

Conventional frozen cryopreservation is currently the gold standard for cardiovascular allograft preservation. However, inflammation and structural deterioration limit transplant durability. Ice-free cryopreservation has already demonstrated matrix structure preservation combined with attenuated immune responses in published large animal studies. In this study the mechanisms of this diminished immunogenicity in vitro were explored. Human aortic tissue punches (8 mm diameter) were frozen according to either the conventional or the ice-free cryopreservation protocol with random allocation. Tissue punches for conventional frozen cryopreservation was done by incubating each punch in 5 mL DMEM containing 10% human albumin and 10% DMSO for 1 h on ice. The punches were then transferred to cryotubes and control rate frozen at 1°C/min to -80°C and stored vapor phase nitrogen for at least 1 month. Ice-free cryopreservation of tissue punches was achieved by incubating each punch in 5 mL of an 83% cryoprotectant solution designated VS83 (Euro-Collins solution containing 4.65 mol/L formamide, 4.65 mol/L DMSO, and 3.31 mol/L 1,2 propanediol) for 1 h at room temperature. The punches were then placed in cryotubes with VS83 and stored at -80°C for at least 1 month. Punches were rewarmed and washed and employed in experiments. First, we characterized factors released by human aortic tissue after freezing and ice-free cryopreservation. Secondly, we analyzed co-cultures with human peripheral blood mononuclear cells, purified monocytes, T cells and monocyte-derived macrophages to examine functional immune effects triggered by the tissue or released cues. Ice-free tissue exhibited significantly lower metabolic activity and release of pro-inflammatory cytokines (IL-6, MCP-1 and IL-8) than frozen tissue, but surprisingly, more active transforming growth factor β. IL-10 was released in equal amounts from both tissues. Due to reduced cytokine release by frozen tissue, less monocyte and T cell migration was detected in a chemotaxis system. Moreover, only cues from frozen tissue but not from ice-free tissue amplified αCD3 triggered T cell proliferation. In a specifically designed macrophage-tissue assay, we could show that macrophages did not upregulate M1 polarization markers (CD80 or HLA-DR) on either tissue type. There was a trend towards an increase in the M2-type marker CD206 in co-cultures with either matrix type. In conclusion, ice-free cryopreservation selectively modulates tissue characteristics and thereby attenuates immune cell attraction and activation. Ice-free cryopreservation may have benefits in terms of preventing adverse immune responses to natural or engineered tissue matrices.

6. Michael Buckenmeyer

Department of Bioengineering, University of Pittsburgh (1); Department of Obstetrics, Gynecology and Reproductive Science, Magee Womens Research Institute (2); Department of Developmental Biology, University of Pittsburgh (3); Department of Microbiology & Molecular Genetics, University of Pittsburgh (4)

Female cancer patients have a significant risk of losing reproductive function due to harsh treatments. The current options for fertility preservation are limited to (1) re-implantation of cryopreserved ovarian tissues or (2) follicle isolation for in vitro maturation (IVM). To date, there has been an increasing number of successful orthotopic transplantation procedures, resulting in greater than 130 live-births; however, the efficiency of this method remains low, with live-birth rates ranging from 23-36%. To improve these outcomes, we have developed a bioactive tissue-specific hydrogel from decellularized porcine ovarian tissues, which could provide an alternative biomaterial to support follicle maturation.

Porcine ovaries were decellularized using a series of detergents then characterized using histology to confirm the removal of cells and the preservation of ovarian structures. PicoGreen assays were used to provide a quantitative assessment of residual dsDNA and gel electrophoresis was used to measure remaining DNA fragments. Ovarian-specific hormones and growth factors were quantified using ELISA assays. Extracellular matrix (ECM) components were analyzed using GAG and hydroxyproline assays. Ovarian ECM proteins were identified using immunohistochemistry. Decellularized tissues were lyophilized, milled into a fine powder then enzymatically digested. Hydrogels were formed at 37°C via a physical crosslinking after neutralizing the digested ECM. The hydrogel mechanical properties and ultrastructure were assessed using rheology and scanning electron microscopy (SEM). Secondary ovarian follicles were mechanically isolated from 16-day old C57BL6/j female mice using insulin syringes. Healthy follicles were selected and encapsulated in ovarian hydrogel (OECM) droplets then cultured for six days in growth media. Bright field images were used to assess follicle diameter and morphology.

Histological analyses verified decellularization, showing an absence of cell nuclei while maintaining the structural integrity of individual follicles. A PicoGreen assay supported these findings, resulting in a 91% removal of dsDNA. Ovarian-specific hormones, growth factors, and ECM proteins were all preserved after decellularization. The OECM hydrogel storage and loss modulus increased in response to higher ECM concentration and SEM analysis demonstrated a highly porous and fibrous ultrastructure. Ovarian follicles encapsulated in OECM droplets showed an increase in follicle diameter and maintained normal morphology after six days in culture. Our results indicate that OECM hydrogels can support follicle growth for at least six days in culture suggesting that this novel biomaterial could be used for IVM to obtain mature oocytes for fertilization and may be utilized as a delivery vehicle for follicle transplant.

7. Lia Campbell

Preservation and Maintenance of Engineered Tissue Construct Function

Lia H. Campbell; Greg Wright; Lindsay Freeman; Kelvin G.M. Brockbank

Tissue Testing Technologies LLC

Now that human tissue engineered models are certified for use in cosmetic testing as a replacement for animal testing, a solution for the storage and shipment of these constructs is a growing commercial problem. Currently epithelial tissue models (usually basal layer to apical surface) such as EpiDerm and EpiOcular (manufactured by Mattek Corp) are processed on inserts in 24 wells plates in ”made-to-order” batches with a 2 week processing-to-shipment time. Tissue models have a limited shelf life and can be recalled if they do not pass quality checks conducted post shipment. A solution to the logistical concerns from “made to order” processing is cryopreservation of the constructs with long term storage in vapor phase nitrogen. Simply freezing the constructs using conventional cell freezing methods does not work due to the damaging effects of ice formation. Early freezing experiments using DMSO only yielded ~2% viability of the constructs. Our alternative strategy, was the development of a cryopreservation method known as ice-free. Ice-free vitrification avoids ice-induced damage with retention of cell viability and matrix structure. A multistep addition and removal protocol is used to add high concentrations of cryoprotectant formulations that are required for inducing vitrification and avoiding ice formation. Experiments were performed using different cryopreservation containers including vials and multiwell plates. After rewarming, the constructs were assessed for viability using alamarBlue for several consecutive days. Implementation of ice-free vitrification with ≥55% cryoprotectants demonstrated significant improvement and viability after preservation. Protocol optimization further improved construct viability to ~80% consistently using the alamarBlue assay. In addition to the alamarBlue assay, more experiments have been performed examining tissue viability using alternate assays such as the MTT assay. The MTT assay also measures metabolic activity and it is the method of choice for assessment of toxicity in these constructs. Functional assays were also performed and includedmeasurement of Triton X-100 cytotoxicity and IL-1α production. Investigations are ongoing to evaluate other vitrification solutions to further improve construct viability, yielding viability results in the 90% range. Overall vitrification is a viable method for preserving human tissue engineered constructs. These results demonstrate the exciting potential of ice-free vitrification for cryopreservation of tissue engineered constructs and the potential for changing current practice for construct storage and distribution paradigms.

8. Erica Castillo

A Mechanically Tunable Hydrogel for Investigating the Mechanobiology of hiPSC-CMs Cell-matrix Interactions

Cardiomyocytes differentiated from human induced pluripotent stem cells (hiPSC-CMs) are a promising platform for developmental, disease, and pharmacological studies. The extracellular matrix (ECM) forms part of the cellular microenvironment and has been shown to critically modulate mechanotransduction through cell-matrix interactions. However, the standard ECM used extensively for hiPSC-CM culture is Matrigel, a complex, biological-derived material with batch-to-batch variations. Matrigel introduces confounding effects to a controlled study of cell-matrix interactions. Cell-ECM interactions can be quantified by measuring contractility and focal adhesions. Ligand density, cell spread area, and substrate stiffness have been shown to influence traction forces and formation of focal adhesions in other cell types. In the present study, a lift-off micropatterning technique allows for precise and repeatable protein patterning on the surface of a polyacrylamide hydrogel. Elastin-like proteins (ELPs) are engineered, recombinant, matrix-mimetic proteins that allow for independent tunability of ligand type, ligand density, and matrix stiffness. Substrate stiffness is tuned by altering the polyacrylamide hydrogel composition, while cell aspect ratio, ligand type, and ligand density are tuned through use of the patterned ELP. Contractility measurements are made by analyzing fiducial markers distributed throughout the hydrogel. We hypothesize that we can study the impact of ligand density, cell spread area, and substrate stiffness on hiPSC-CM force production and focal adhesions. Preliminary results show average force production by hiPSC-CMs of 0.07 uN and 0.04 uN on Matrigel and ELP-RGD, respectively. hiPSC-CMs on ELP-RGD show a total force of 0.006 uN, increasing to 0.06 uN as the length:width cell aspect ratio increases from 4:1 to 7:1.

9. Robert Coyle

Glucose Diffusion as the Limiting Factor in hADSC Spheroid Viability under Ischemic Conditions

Bioengineering, Clemson University (1); Regenerative Medicine, Medical University of South Carolina (2)

Human adipose derived stem cell (hADSC) spheroid microtissues have shown great potential as a method for treating ischemic injury. Properties of these constructs, such as their secretion of pro-survival paracrine factors and their resistance to hypoxia, suggest their useful application in regenerating sites of ischemic injury in muscle tissue. However, the harsh conditions of the ischemic environment and the avascular nature of hADSC spheroids results in limited viability of the tissue constructs. We aimed to show that this decrease in viability is a function of both spheroid diameter and the availability of metabolic substrates. To this end, we developed an in vitro ischemic model that allowed for control of metabolite concentrations and exposure time for varying spheroid diameters. Through the use of TUNEL staining, we were able to show that under ischemic conditions, there was a correlation between increased diameter and decreased spheroid viability. However, it is possible to mitigate the effects ischemic and hypoxic conditions through maintaining high concentrations of glucose, but not through high levels of oxygen. Through COMSOL mathematical modeling, we were able to show a correlation between glucose availability and spheroid viability. These results help explain the high viability recorded after exposure to conditions of low oxygen and high glucose concentrations. The modeling also supports results showing higher viability of small hADSC spheroids relative to large hADSC spheroids as a function of glucose availability. In this study, we were able to demonstrate that under stressful conditions, decreasing spheroid size and maintaining high levels of glucose were sufficient to mitigate the detrimental effects of ischemia. These results may impact future strategies for transplanting hADSCs through an optimized spheroid diameter and the co-delivery of metabolic substrates.

Cell-extracellular matrix (ECM) interactions transduce mechanical and biochemical signals that regulate epithelial morphogenesis. Understanding these interactions has been a major goal for biomaterials scientists in order to engineer materials that can recapitulate complex ECM-mediated cellular responses.

The Zent Lab has established the use of 3D type I collagen gels for the culture of different kidney-associated epithelial cells (e.g. inner medullary collecting duct, IMCD) that recreate the normal and diseased tubular morphogenetic developmental programs observed in the organism. However, the inability of this material to decouple mechanical and biochemical properties limits its use for the understanding of the independent contributions of matrix properties to epithelial morphogenesis. Therefore, there is a significant need for a biomaterial matrix that can recapitulate epithelial tubular morphogenetic programs while overcoming this limitation.

The objective of this project is to engineer a synthetic, biofunctionalized hydrogel platform that allows the understanding of the independent contributions of matrix properties to epithelial tubule morphogenesis. Tunable, cell-encapsulating hydrogels were formed from a four-armed, polyethylene glycol macromer having maleimide groups at each terminus (PEG-4MAL) that are conjugated with cysteine-containing adhesive and crosslinking peptides.

We engineered a fully defined PEG-4MAL hydrogel that recapitulates mouse IMCD tubular morphogenetic program in vitro without the use of type I collagen gels. Both mechanical and biochemical properties of the synthetic ECM were important to epithelial tubulogenesis, consistent with previous work showing that human epithelial organoid generation is controlled by ECM elasticity and adhesive peptide type in 3D cultures (Cruz-Acuña and Quirós et al., 2017). The independent contributions of physicochemical matrix properties on IMCD cell morphogenetic program were evaluated by assessing cell viability, proliferation, cluster size, and a comprehensive study of tubule phenotype (e.g. tubule number, length and intersections). We demonstrated that PEG-4MAL hydrogel serves as a platform to characterize different matrix property-dependent tubular phenotypes which helps understand the independent contributions of matrix properties to healthy and diseased tubular morphogenesis.

Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University (1); Division of Cardiology, Emory University School of Medicine (2); Division of Nuclear Medicine and Molecular Imaging, Emory University Department of Radiology and Imaging Sciences (3); MR Research Division, Emory University Department of Radiology and Imaging Sciences (4)

Peripheral artery disease (PAD) remains a significant age-related medical condition with limited treatment options in late stages. Cell therapy has emerged as a promising approach to regenerate vasculature in PAD patients; however, clinical trials have been inconclusive. A large animal preclinical PAD model has not been standardized, and non-quantitative approaches have been taken to characterize and assess efficacy. Alginate-encapsulated mesenchymal stromal cells (MSCs) have shown promise in promoting regeneration in small animal PAD models through paracrine factor secretion and increased retention and viability. This project aims create and sophisticatedly characterize a porcine PAD model and visualize the viability, retention, and distribution of encapsulated MSCs in PAD models.

A swine pilot study was conducted to create a PAD model using a ligation/excision approach. Gait analysis with a walkway system was performed pre- and post-ligation to subjectively quantify functional deficits. Arterial spin labeling (ASL) magnetic resonance imaging (MRI) was performed at various occasions post-ligation to measure microvascular perfusion in hind limb skeletal muscle during cuff-induced reactive hyperemia. Additionally, porcine MSCs were transduced with a custom lentiviral vector to express a reporter gene for visualization with positron emission tomography (PET).

Automated gait analysis detected significant abnormalities at Day 1 post-ligation that returned to baseline at Day 6. ASL MRI successfully quantified microvascular perfusion in hind limb skeletal muscle; however, no trend was observed between time points. Angiography at Day 43 detected robust collateral formation revascularizing the ischemic limb. Porcine MSCs could be engineered to express a functional reporter protein that enables uptake of a radioactive tracer in vitro.

The pilot porcine PAD model characterization study failed to produce a sufficient ischemic time window to test encapsulated MSC efficacy. However, the surgical approach taken to create the model was feasible. Gait analysis with a walkway system and skeletal muscle perfusion measurements with ASL MRI will be valuable tools moving forward to quantitatively assess the efficacy of alginate-encapsulated MSCs in a more severe large animal PAD model. Engineered porcine MSCs show promise for detection in vitro via PET in both small and large animals to non-invasively assess viability, retention, and distribution over time.

Department of Plastic Surgery, University of Pittsburgh (1); Department of Bioengineering, University of Pittsburgh (2); McGowan Institute for Regenerative Medicine (3); Department of Neuroscience, University of Pittsburgh (4); Axogen Corporation (5)

Peripheral nerve injuries are common and account for 2-5% of patients entering Level I trauma facilities annually and can be caused by physical trauma such as military blast injuries or tumor excisions. The gold standard to repair these injuries is autografting, which involves harvesting a portion of non-essential sensory nerve and transplanting it into the gap. Complications of autografting include neuroma formation, loss of motor and sensory function at the donor site, increased operative time, risk of infection, and lack of sufficient donor tissue. Our laboratory has demonstrated successful regeneration in non-human primates using a poly(caprolactone) (PCL) nerve conduit embedded with glial cell line-derived neurotrophic factor (GDNF) as well as decellularized nerve allografts. Axomax™ combines the PCL/GDNF conduit with a decellularized nerve inserted into the inner lumen of the guide. This provides an extracellular structure for cells to repopulate and a potent Schwann cell recruiter, GDNF, to enhance and stimulate nerve regeneration. The purpose of this study was to determine the efficacy of Axomax™ in treating nerve regeneration in a rodent model.

A 1.5cm critical defect was created on the sciatic nerve of male Lewis rats. The gap was repaired with the following treatment groups: Axomax™, reverse polarity autograft, decellularized nerve allograft, or the composite guide without GDNF. Each treatment group had n=8. Treated and native nerves were explanted at 6-weeks postoperatively along with the gastrocnemius muscle, which is innervated by the sciatic nerve. Treated and native muscle were weighed. Nerves were fixed and stained with Masson’s Trichrome, S100 and neurofilament. Muscles were stained for dystrophin. Gastrocnemius Muscle Weight Ratio (GWR) was calculated. Cumulative GDNF release data was gathered.

The GWR for Axomax™ was comparable to autograft, and was significantly higher than the empty composite guide and decellularized nerve alone. More fibrous tissue formation was observed in the empty composite guide. The Axomax™ composite guide decreased muscle atrophy at 6-weeks in rats and provides a sustained release of GDNF, promoting nerve regeneration. Decreasing muscle atrophy may result in better functional outcomes. Future studies include adding physical therapy during the recovery phase to improve distal muscle function.

Because of their ability to secrete cytokines and growth factors that modulate the innate immune response, mesenchymal stem cells (MSCs) have been used in several clinical trials to reduce symptoms and promote tissue healing in multiple disease states (Squillaro et al. Cell Transplant, 2016). Yet despite their clear therapeutic potential, MSC-based therapies have demonstrated a range of efficacy. This inconsistency has largely been attributed to variability of the secretome among MSCs from different donors cultured under varying conditions (Mendicino et al. Cell Stem Cell, 2014). Thus, there is a need to characterize the secretome and predict the potency of MSCs from individual donors before they are used in clinical trials in order to achieve consistent therapeutic outcomes. In this work, we used a multivariate secretome characterization of MSCs cultured under different physical conditions in order to distinguish between MSCs from individual donors.

To characterize the secretomes from individual donors, we analyzed their conditioned culture media for over 40 different immunomodulatory factors. MSCs isolated from bone marrow aspirates from 4 donors were either separately formed into 500-cell aggregates and placed in suspension culture or plated at 13,000/cm2 in static culture. After 4 days in culture, conditioned media was collected and quantified for various immunomodulatory factors using magnetic bead-based cytokine assay kit (HCYTMAG-60K-PX41, Millipore), individual PGE-2 and TSG-6 ELISAs (R&D Systems) and a previously-established IDO enzymatic activity assay (Agaugué et al. J Immunol 2006). Secretome data was normalized to the total number of cells in culture, determined by an automated cell counter (Countess II, Invitrogen).

Of the 43 screened immunomodulatory factors, 18 of the factors were found to be secreted in detectable amounts by MSCs from all donors in both monolayer and aggregate cultures. Formation into 500-cell aggregates increased the secretion of the majority of detectable immunomodulatory factors in 3 of the 4 cell donors. Although no secretory differences were found between cell donors in monolayer culture, formation of MSCs into aggregates revealed differences in the secretion of 10 of the 18 detectable factors (p<0.05). Applying multivariate statistical analysis (partial least squares discriminant analysis) created a model from the secretome data that could significantly discriminate among the secretory profiles of MSC aggregates from all but 2 donors (p<0.05).

This study demonstrates that secretome analysis can be used to characterize MSC donor-to-donor variability. Additionally, this work shows that amplification of secretome through cellular aggregation can increase the sensitivity of this method to assess donor variability. Because MSCs are thought to act through secreted factors, in the future, secretome analysis and modeling may become an important metric in determining differences in cell potency.

Department of Biomedical Engineering, Parks College of Engineering, Aviation, and Technology, Saint Louis University (1); Department of Orthopedic Surgery, Saint Louis University (2)

Musculoskeletal injuries are among the most common and frequently disabling injuries sustained by athletes and soldiers. Most of these injuries involve volumetric muscle loss (VML), defined as the as the surgical or traumatic loss of muscle tissue with resultant functional impairment. While skeletal muscle is remarkably regenerative, VML injuries are irrecoverable in humans and animal models due to the complete loss of indispensable regenerative elements such as basal lamina and resident satellite cells. Currently, there are no approved therapies for the treatment of muscle tissue following trauma, presenting a significant opportunity to develop tissue engineered scaffolds for muscle tissue regeneration.

To improve regeneration of skeletal muscle, we have developed biomimetic sponges composed of collagen, gelatin, and laminin (LM)-111 that were crosslinked with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC). Collagen and LM-111 are crucial components of the muscle extracellular matrix and were chosen to impart bioactivity whereas gelatin and EDC were used to provide mechanical strength to the scaffold. Morphological and mechanical evaluation of the sponges showed porous structure, water-retention capacity and a compressive modulus of 590kPa. In vitro testing revealed that compared to pure gelatin sponges, the biomimetic sponges supported greater C2C12 myoblast infiltration, myokine (VEGF, IL-6, and IGF-1) production and myogenic marker (MyoD and myogenin) expression.

The biomimetic sponges were implanted in a mouse model of VML. At 2 weeks post-injury, the biomimetic sponge treated VML injured muscles showed constructive remodeling at the site of injury with the elevated presence of satellite (Pax7+), endothelial (CD31+) and inflammatory (F4/80+) cells compared to untreated VML injured muscles. The sponge treated muscles showed several small diameter myosin+ myofibers in the defect region and a higher quantity of centronucleated myofibers per muscle section. In support, the protein expression of MyoD and myogenin was also higher on the sponge treated injured muscles. However, the expression of heat shock protein (HSP)-70, a marker of cellular stress was lower with sponge treatment. Taken together, these results suggest that implantation of the biomimetic sponges is able to promote myogenic activity in the VML injured muscles. Future studies will evaluate the extent to which biomimetic sponges can improve muscle regeneration and force production at one-month post-VML injury.

15. Jonathan Griffin

J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, Gainesville, FL

Spinal cord injuries (SCIs) affect approximately 285,000 people in the US alone and often result in para- or tetraplegia. Currently there is no approved treatment for SCI but numerous approaches are being researched. One of the most promising strategies is the use of decellularized biologic scaffolds. These scaffolds are highly desirable as they maintain native extracellular matrix (ECM) protein content and architecture. Scaffolds can be useful for various applications in regenerative medicine such as delivery of therapeutics and providing topographical cues. However, to prevent unwanted immune reactions, it is important to remove cellular components. SCIs commonly cause cavitation at the site of contusion, making minimally invasive injectable treatments advantageous. Cavity filling, thermally gelling scaffolds have been previously investigated in the Schmidt Lab (in collaboration with Dr. Mary Bunge) and were shown to improve regeneration and functional recovery in vivo when injected along with Schwann cells. These injectable scaffolds were developed by digesting sciatic nerves that had been decellularized via washing tissues in chemicals to lyse and remove cellular components. Although this approach facilitates the removal of cellular components, the harsh conditions cause disruption to ECM proteins (laminin and collagen) that are critical for axonal regeneration. Cell lysis results in ruptured cell membranes, which leads to the release of immunogenic intracellular elements into the extracellular environment. This undesirable aspect of current decellularization methods likely makes complete removal of cellular elements more difficult. An alternative approach to chemical detergents is induction of apoptosis. Apoptotic cells undergo organized fragmentation into small membrane bound bodies that become detached from the ECM. This method does not require the use of chemical detergents and allows for shorter wash times, increasing preservation of ECM. Our present goal is to develop an injectable, thermally gelling scaffold derived from rat sciatic nerve decellularized via apoptosis rather than harsher previously employed methods. Preliminary data from the Schmidt Lab suggests decellularization via apoptosis can adequately remove cellular content while preserving crucial ECM proteins. By tuning digestion conditions such as temperature, tissue concentration, digest duration and acid type, recent attempts to thermally gel decellularized nerves have been successful. In future experiments, we aim to thoroughly characterize our scaffolds. Both local and bulk mechanical properties, protein content and scaffold microarchitecture will be investigated. Looking even further ahead, we plan to use these scaffolds to establish localized and controlled delivery of therapeutics to stimulate axonal outgrowth and functional recovery in a rat SCI model.

Duchenne muscular dystrophy (DMD) is a devastating genetic disorder that affects approximately 1 in 3,500 males. A leading cause of death in DMD is diaphragm muscle deterioration, and current respiratory care, such as mechanically assisted ventilation, remains palliative. A therapeutic strategy targeting the diaphragm muscle to restore the respiratory capacity is in a critical need. DMD is caused by the absence of dystrophin, a structural protein that provides support between the muscle fiber cytoskeleton and the extracellular matrix. Restoration of dystrophin through muscle satellite cell transplantation improves the muscle function, but direct cell delivery is limited by sub-optimal survival and engraftment. Furthermore, cell delivery strategies designed to target the anatomically deep-seated and dimensionally thin diaphragm muscle have not been developed. The objective of this work is to engineer a synthetic matrix to facilitate the delivery and engraftment of muscle satellite cells into the dystrophic diaphragm muscles. We engineered a synthetic matrix that supports primary satellite cell survival, proliferation, and differentiation using hydrogels based on PEG-4MAL macromers. Satellite cells cultured in the RGD-presenting hydrogels formed significantly larger MyoD+ 3D myogenic colonies after 6 days of culture compared to colonies formed in RDG, YIGSR, and C16-presenting hydrogels (p<0.0001). While no differences in EdU incorporation were observed, cell survival was significantly higher in the RGD-presenting hydrogels compared to RDG-presenting hydrogels (p<0.05), suggesting that RGD-presenting hydrogels promote cell survival and subsequently stimulate the formation of large myogenic colonies. Myogenic colonies formed in the RGD-presenting hydrogels exhibited significantly lower cell packing density compared to RDG, YIGSR, and C16-presenting hydrogels (p<0.0001), indicating cellular migration. When primed for differentiation, the cells in the RGD-presenting hydrogels formed significantly higher multinucleated myotubes compared to the cells in the RDG-presenting hydrogels (p<0.0001). The optimal material stiffness (G’ ~175 Pa) and mesh size (30 nm) of the hydrogel was also determined by modulating the PEG-4MAL macromer density to further enhance the satellite cell function in 3D. We further developed a delivery strategy to firmly integrate the engineered hydrogel to the inferior surface of the dystrophic diaphragm. Fluorescently-labeled hydrogel delivered to the diaphragm remained localized to the site of delivery, whereas fluorescently-labeled, uncrosslinked hydrogel precursor solution resulted in a non-specific distribution to other internal organs including the large intestine, stomach, and liver. Finally, GFP+ satellite cells delivered to the diaphragm using the engineered hydrogel survive, proliferate, and engraft in vivo.

Satellite cells are myogenic stem cells that play a critical role in skeletal muscle repair, dormant in healthy muscle they activate upon injury with increased proliferation and differentiation into myoblasts. Since reestablishing vascular supply following injury is an important part of muscular repair, we hypothesized that satellite cells might also play a role in regulating vascular growth through paracrine signaling. This is of particular interest to us in the context of peripheral artery disease where ischemic tissue damage is a major complication and the generation of a robust collateral network improves the prognosis. Using a murine model of hind limb ischemia, we showed that satellite cells increased 3.4 fold (p<0.01) in response to ischemia. We used a co-culture system to demonstrate that satellite cells led to a 3.5 fold increase in smooth muscle migration (p<0.0001) and a 2.8 fold increase in endothelial cell migration (p<0.0001). These results demonstrate that the satellite cells produce paracrine factors which drive both smooth muscle and endothelial cell migration which is required for the development of collateral vessels. To test the potential therapeutic capability, alginate encapsulated satellite cells were delivered in the hind limb ischemic model. We found the encapsulated cells were viable for up to 2 weeks and mice that received satellite cells had significantly increased perfusion (28%, p<0.05) at 2 weeks as measured by Laser Doppler imaging and a 1.9 fold (p<0.5) increase in capillaries and small vessels as measured by histological staining. To examine the role of satellite cells in a physiological setting and determine if they are critical to robust recovery in our model, we used a Cre-Lox system in which recombination results in the production of diphtheria toxin a (DTA) to deplete Pax7 specific cells. Mice which lacked satellite cells had decreased perfusion by Laser Doppler imaging (p<0.05) and capillary density compared animals with intact satellite cells. A cytokine array and genomics analysis show that several factors related to angiogenesis and cell migration are upregulated in satellite cells in response to ischemia. In conclusion our studies show that satellite cells increase in response to ischemia, produce paracrine factors that increase vascular cell migration in vitro, and lead to increases in perfusion in vivo. We believe these results demonstrate the critical role satellite cells play in collateral vessel formation and may be a potential new therapeutic approach for treating peripheral artery disease.

18. Martin Haschak

McGowan Institute for Regenerative Medicine, University of Pittsburgh (1); Department of Bioengineering, University of Pittsburgh (2); Department of Biological Sciences, University of Pittsburgh (3); Department of Obstetrics, Gynecology, and Reproductive Sciences, University of Pittsburgh (4)

Clinical interventions following a myocardial infarction (MI) event are limited in their efficacy due to the limited regenerative capacity of post-mitotic, adult mammalian cardiomyocytes, which are depleted in large quantities during ischemic cardiac injury. However, potent cardiac regeneration in neonates and limited cardiomyocyte proliferation in aged individuals following MI has been observed. This observed neonatal cardiac regeneration was not holistically dependent on non-terminally differentiated cardiomyocytes but was also dependent on the macrophage populations present in the tissue. Cardiac macrophages are derived from three unique sources- the extraembryonic yolk sac, fetal liver, and bone marrow- and colonize the cardiac tissue during distinct periods of development. Interestingly, a recent study demonstrated the ability to recapitulate the neonatal regenerative response in adult cardiac tissue through the selective recruitment of yolk sac-derived macrophages to the infarct site. However, the factors governing the maintenance and recruitment of these macrophages in aged individuals remains unclear. The local microenvironment composed of extracellular matrix (ECM), growth factors, chemokines, and numerous additional soluble factors plays a substantial role in determining macrophage development and phenotype. Recently, several tissue-specific macrophage niches have been identified as essential determinants of macrophage phenotype, independent of macrophage developmental origin. Thus, this study sought to examine the potential role cardiac ECM aging plays in altering macrophage phenotype and functionality. A decellularization protocol was optimized to isolate murine cardiac ECM. Biochemical and DNA quantification assays were performed to ensure sufficient decellularization. Bone marrow derived macrophages isolated in culture were exposed to either young or aged cardiac ECM degradation products. Following 24 hour ECM exposure, macrophage nitric oxide production and gene expression levels were assessed at baseline and following exposure to canonical M1 or M2 polarizing cytokines. Macrophages exposed to aged cardiac ECM degradation products exhibited increased nitric oxide production compared to young ECM-exposed groups, a functional response exhibited in pro-inflammatory macrophages. Additionally, qRT-PCR analysis revealed several alterations in gene expression following ECM exposure with subsequent cytokine polarization. These results indicate that macrophages exhibit differential responses to young and aged ECM degradation products derived from cardiac extracellular matrix in vitro.

Department of Biomedical Engineering, Parks College of Engineering, Aviation, and Technology, Saint Louis University (1); Department of Orthopaedic Surgery, Saint Louis University (2); Department of Biology, Saint Louis University (3)

Clinically, critical-size defects are defined as a defect twice the size of the bone’s diameter which will not heal spontaneously. The current gold standard for treatment involves the use of a bone graft; however, there is limited availability, high cost, potential for infection, and donor site morbidity. Cryogel scaffolds are unique tissue-engineered constructs formed at sub-zero temperatures. When thawed, the resulting structure is macroporous, sponge-like, and mechanically durable. Due to these properties, cryogels have excellent capability for the treatment of critical-size defects.

Chitosan-gelatin cryogels were previously incorporated with various forms of hydroxyapatite to expediate the process of mineralization during bone regeneration. It was demonstrated that these cryogels doped with 5% bone-char (BC) had high pore interconnectivity and full cell infiltration by day 28. Additionally, despite the high-density of BC the cryogels were able to swell to their full potential within two minutes of being immersed in water, demonstrating their potential to fill a bone defect site. Thus, a cryogel incorporated with BC was found to be a cost-effective bone graft substitute by increasing mineralization at the wound site.

In a separate study, silk fibroin cryogels were incorporated with various concentrations of Manuka honey (MH), a positive agent in bacterial inhibition and mediator in decreasing pH at the wound site. These cryogels possessed appropriate porosity, durability, and swelling kinetics. Additionally, a sustained release of glucose was observed along with clearance of S. aureus and decreased adhesion of the bacteria. Overall, the addition of 5% MH produced a cryogel that could inhibit bacterial growth, while still maintaining the desired structural characteristics for bone regeneration.

Cryogels incorporated with both additives were characterized to determine both the effect on their spongy, macroporous structure and the retention for bacterial clearance and mineralization. Initial testing found that 2.5% of both additives was sufficient, deeming this concentration ideal for stem cell studies. While we have demonstrated in vitro success with these materials and cryogels to serve as a template for bone regeneration, our current work is focused on using the cryogel to stimulate stem cell populations and promote healing in vivo.

At the time of submission we are focused on the differentiation of mesenchymal stem cells (MSCs) towards a bone lineage when in contact with our doped cryogels. Analysis methods have included alizarin red stain and microCT for quantification of mineral deposition, as well as assays for markers of bone differentiation. Additionally, we are piloting a murine cranial defect model with these cryogels. While this data is pending, preliminary work has proved fruitful and demonstrates the efficacy of these scaffolds in promoting bone regeneration.

Pelvic organ prolapse, a disorder in which the muscles of the pelvic floor are weakened over time, affects over a million women each year in the United States. A quarter of these women undergo a reconstructive procedure, increasingly using polypropylene mesh as mechanical reinforcement to the pelvic floor. However, the number of complications such as chronic pain and mesh erosion/exposure in women with vaginal mesh implants were reported at rates as high as 10-20 %. This indicates a limited understanding of the host response to mesh in vaginal tissue and strategies to reduce these complications.

Utilizing a novel surgical technique in New Zealand white rabbits, we implant mesh using the “gold standard” abdominal sacrocolpopexy procedure and evaluate changes in the immunologic response at early (14 days) and tissue remodeling outcomes at late stages (90 and 180 days) of implantation. The procedure begins with an initial hysterectomy removing the uterus followed by creating space along the vaginal wall on both sides between the bladder and the rectum. Two 3 x 10 cm2 pieces of mesh are secured along both sides of the vaginal wall. The remaining flaps at the top are then secured to a ligament in the sacral/lumbar space, creating the support to the pelvic organs. Upon closing the incision, a partial thickness defect is made in the abdominal wall, and mesh is implanted inside to repair the abdominal muscle. Both of these implantations of mesh allow for the assessment of the immune response in the pelvic area (relevant for prolapse patients) and in the abdominal area (relevant for translation from hernia repair).The mesh-tissue complex was removed from each rabbit and processed for histological staining as well as immunolabeling of immune cells, such as macrophages. Extracellular matrix protease assays and mechanical integrity of the tissue also evaluate the overall inflammatory response associated with each implant.

The results of this study show that implants into vaginal tissues elicited an increased host inflammatory response at 14 days as compared to those in the abdominal wall. However, at chronic time points the inflammatory response in the vagina was reduced as compared to that in the abdominal cavity. The present study also demonstrates the scale-up of a previous methodology for a nanoscale coating. We present a nanometer thickness, tunable, and uniform coating capable of releasing bioactive IL-4. We evaluated the biological functionality of the coated mesh via bioactivity studies and in vivo implantation. An ideal mesh would provide mechanical support to the pelvic floor while decreasing the inflammatory response and increasing integration with the surrounding native tissue.

21. Moriah Johngrass

Development of a Clinically Relevant Xenograft Model for Localized Fat Graft Tumor Suppression

Approximately 1 in 8 U.S. women will develop invasive breast cancer over the course of her lifetime, many of which will result in mastectomy. Reconstruction methods based on aspirating and reinjecting fragmented adipose tissue (autologous fat transfer) are currently used clinically to treat all breast cancer patients in both a primary reconstruction method and to improve outcomes in patients who have undergone flap or implant procedures. Even after a successful reconstruction, approximately 10 to 15% of patients with invasive breast cancer will develop a clinically isolated local recurrence. Our long-term goal is to develop an injectable, tumor suppressive autologous tissue technique that combines current AFT reconstruction techniques with chemotherapeutics, generating natural and viable tissue that also reduces tumor recurrence. We hypothesize that mixing chemotherapeutics encapsulated in degradable microspheres with autologous adipose tissue prior to tissue injection for reconstruction will be an effective method for reducing tumor growth.

To test this hypothesis, we have developed a novel mouse model using an established human mammary gland metastatic tumor cell line, MCF-7. This cell line was chosen because they are positive for estrogen receptors (ER+). Prior to testing our combined therapy, the animal model was first developed through a staged- approach to validate performance of controls. In our first study, we determined the required MCF-7 cell dose for detecting differences in tumor size at our desired study endpoint, which was 6 weeks. 15 female NOD-SCID mice were separated into three groups (n=5 per group) and four mammary pads were each injected with 1.0 x 105. Mice were sacrificed at 10 weeks and tumor volume was calculated. Results from this study showed that 105 cells produced small tumors at 6 weeks (µ=10 ± 10.8mg), which did not meet outcome requirements. Therefore, a second study was conducted in which we increased the cell dose to 106 cells. Positive and negative controls used in this study included estradiol delivered through pellets (0.18 mg/kg) that were implanted subcutaneously in the neck 24 hours before MCF-7 injection. Paclitaxel (taxol), a clinically established FDA-approved drug used to treat breast cancer since 2005, was selected for proof of concept studies and was thus first evaluated through standard of care delivery.

Observational results showed that this cell dose and treatment regime successfully generated significant differences in tumor size at the completion of the study. Our next step will be to validate adipose tissue viability after the inclusion of encapsulated paclitaxel microparticles. The conclusion of this study will bring us close to showing efficacy of localized ATF chemo release in suppressing tumor growth.

As the second leading cause of death in adults over the age of 60, stroke affects approximately 15 million people every year, resulting in 6 million deaths worldwide. Recent rodent studies suggest mesenchymal stem cell extracellular vesicles (MSC EVs) may serve as an effective therapeutic. In this study, the efficacy of novel EVs derived from human neural stem cells (NSCs) were tested in a translational pig ischemic stroke model. We hypothesized NSC extracellular vesicles (NSC EVs) would result in decreased cerebral infarct volume, reduced hemispheric swelling, conservation of white matter integrity, and improved diffusivity at acute and chronic time points.

Collectively, these results supported our hypothesis indicating NSC EV treatment led to decreased infarct volume, reduced hemispheric swelling, improved white matter integrity in the corpus callosum, and preserved diffusivity in ischemic regions. With these improvements in both the acute and chronic stages of stroke, NSC EV represent a highly practical and translatable therapy that may change the current therapeutic paradigm of stroke in humans.

23. Heidi Kletzien

University of Wisconsin School of Medicine and Public Health, Otolaryngolog,Head and Neck Surgery (1); University of Wisconsin-Madison, Department of Biomedical Engineering (2); University of Wisconsin-Madison, Department of Communication Sciences and Disorders (3)

Regenerative capacity of tongue muscles changes with age. Mechanisms are unknown and may include increased death (apoptosis) of myonuclei and a diminished pool of muscle stem cells. It is also not known if tongue exercise may rescue cellular mechanisms of muscle regeneration in the tongue. The purpose of this study was to determine the effects of age and exercise on underlying cellular mechanisms of tongue muscle regeneration.

Forty-eight young adult, middle-aged, and old F344/BN rats were assigned to tongue exercise or sham-exercise (control) conditions (5 days/wk for 8wk). Following the study period, cross-sections of the genioglossus (GG) and hyoglossus (HG) muscles were immunostained for quantification of apoptotic (TUNEL) and regenerative capacity (Pax7) indices. Western blots for biomarkers of apoptosis and regeneration of whole muscle homogenates will also be performed in the GG and HG.

Preliminary analysis of the GG suggest cell death increases with both age (F2,42=4.5, p=.04) and tongue exercise (F1,42=4.2, p=.02), and that cell death increases in Pax7+ muscle stem cells with aging (F2,42=10.3, p<.001).

Pro-apoptotic processes that preferentially target muscle stem cells may be an underlying mechanism contributing to age-related degeneration of the tongue musculature. Further understanding of cellular mechanisms of muscle degeneration and regeneration are crucial to the development of exercise-based therapies for dysphagia.

24. Leyla Larsson

Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech & Emory University (1); School of Biological Sciences and the Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology (2)

1Institute of Anatomy and Cell Biology, University of Würzburg, Germany

2Cell Therapy Bioprocessing, MilliporeSigma, Bedford, MA, USA

The therapeutic potential of human induced pluripotent stem cells (hiPSCs) is explored in a large array of indications, ranging from acute myocardial infarction to diabetes. The inefficiencies in some current differentiation protocols combined with the large numbers of cells recommended for clinical scale tissue engineering warrant the use of systems that are capable of generating large batches of hiPSCs in a controlled manner. Prior studies have shown the ability to culture hiPSCs in stirred suspension culture, however the culture vessels have mostly been limited to small scale spinner flasks with no monitoring and control of pH and dissolved oxygen (DO).

In this study, hiPSCs were seeded as single cells in 125mL spinner flasks and agitated at a rate that would support aggregate formation. This culture method circumvented the need for a substrate or microcarriers. After growing the aggregates for up to 7 days, they were dissociated into a single cell suspension and cultured for an additional 7 days in a single-use 3L bioreactor, again using an appropriate agitation rate to facilitate aggregate formation. Overall, a 125 fold expansion was achieved after a combined 14 day culture. After aggregate formation and growth in the bioreactor, the hiPSCs were cultured onto a planar culture for characterization assays which included immunocytochemistry of iPSC colonies, flow cytometry, and differentiation toward tissues of the embryonic germ layers. The hiPSCs retained expression of the pluripotency markers and formed tissues of each of the three germ layers. The results demonstrate the potential of hiPSC production in controlled stirred suspension systems that can support the production of large batches of cells for research and clinical applications.

26. Bobby Leitmann

Driftmier College of Engineering, University of Georgia (1); College of Agricultural and Environmental Science

University of Georgia (2); Rhodes Center for Animal Dairy Science, University of Georgia (3)

Mesenchymal stem cell (MSC) therapies have been shown to have great promise in treating inflammatory and autoimmune diseases, but variability in the potency that arises from different MSC donors and the need for biomanufacturing processes to provide large quantities of high quality cells has slowed progress. The current state of the art in predicting MSC potency uses imaging of morphological features to predict immune suppressive properties of the cells. We propose a transformative strategy to use label-free, non-destructive imaging to analyze cell morphology and topography that could be easily integrated in the biomanufacturing pipeline. We treated high potency and low potency donors with interferon gamma and imaged using the label free imaging modality reflectance confocal microscopy. Three dimensional cell morphological features were obtained and analyzed to discern between experimental groups and compared to previously obtained morphological features from labeled images. This has enabled a predictive model to be constructed using the high content imaging data and to determine potency of new untested donors. We expect the label-free, high content imaging analysis to provide insight into MSC potency assessment. Success would result in a reliable mode for quality assessment that can be easily integrated into a biomanufacturing pipeline and provide low cost, high quality cells to the public.

Introduction: Atrial fibrillation (AF) is the most common cardiac arrhythmia. While treatment options for AF exist, many patients cannot be maintained in normal sinus rhythm. Amiodarone is an effective medication for AF but has limited clinical utility due to off-target tissue toxicity. Here, we use a pig model of AF to test the efficacy of an amiodarone-containing poly(ethylene glycol)-based hydrogel.

Methods: Delivery of hydrogels to the cardiac epicardium may be a new treatment strategy for cardiovascular diseases. We recently described a new method to place gels on the epicardium using a minimally invasive procedure.

28. Meghan Logun

Regenerative Bioscience Center, University of Georgia (1); Department of Physics, University of Georgia (2)

Patient prognosis for glioblastoma multiforme (GBM) has remained extremely poor with an average survival of 12 months with best standard of care treatment. Glioma stem cells (GSCs) confer both chemo- and radioresistance, and are similar to neural stem cells (NSCs) in their ability to self-renew and in their dependence on microenvironmental cues for maintenance and progression. GBM tumors rarely metastasize outside of the brain, pointing towards specialized interactions within brain extracellular matrix (ECM) that may contribute directly to both glioma invasion and the harboring of GSC populations. Sulfated chondroitin sulfate glyscosaminoglycans (CS-GAGs) are key components of the NSC niche that regulate NSC maintenance, and elevated levels of oversulfated CS-GAGs in the peritumoral ECM around GBM correlates with poor prognosis. We seek to determine the role of sulfated CS-GAGs in GBM invasion and maintenance of GSC populations using both in vitro methods and a rat glioma model. Human GBM predominantly occurs in the frontal lobe and therefore frontal lobe tumors were induced in rats by inoculating 50k allogeneic F98 cells in media only or in media containing 20 µM of the GAG antagonist “surfen” at stereotaxic coordinates 2mm lateral from midline and 1mm anterior to bregma. Longitudinal magnetic resonance imaging (MRI) was used to track progress of tumor volume, white matter disruption, and angiogenesis over a period of 28 days. Animals were sacrificed at the end of 28 days tumor post-inoculation, and coronal brain tissue sections were immunohistochemically stained at the 28d time point to evaluate tumor associated presence of GSCs (CD133+) and CS proteoglycans (CS-56+ CSPGs). Human patient-derived GSCs cultured with and without 20 µM surfen were examined immunohistochemically for the above described markers along with the neural stem cell marker Sox1 in order to validate surfen mediated inhibition of biomarker co-localization. MRI results confirm the significantly (p<0.05) reduced tumor volumes of surfen treated animals when compared to untreated controls. Immunohistochemical assessment of patient-derived GSCs and tumor tissue sections indicated that surfen treatment reduced the co-localization of CD133+ and CSPGs when compared to untreated controls which demonstrated the strong colocalization of these biomarkers. These observations correlate with the reduced invasion seen in in vivo surfen-treated tumors, suggesting that blocking GSC interactions with extracellular CS-GAGs affects normal GSC phenotype and behavior. Identification of this role for CS-GAGs in GSC behavior would greatly advance our understanding of glioma invasion and contribute to the development of novel therapeutic interventions for malignant glioma.

Department of Biomedical Engineering, Parks College of Engineering, Aviation, and Technology, Saint Louis University

Volumetric muscle loss (VML) is characterized by a loss of muscle accompanied by severe functional impairment and often long-term disability. Clinical therapies currently employed in the treatment of VML are ineffective at regenerating lost muscle and restoring function. We have developed a novel hydrogel composed of fibrinogen (20 mg/ml) and laminin (LM)-111 (450 μg/ml) to promote the regeneration and recovery of VML traumatized muscle. In vitro studies showed that the hydrogels mimic the structural and mechanical properties of the native muscle extracellular matrix (ECM). These hydrogels also enhance C2C12 myoblast proliferation and growth factor production compared to pure fibrin hydrogels. In this study, a VML model was established wherein a full thickness 3 mm biopsy was performed to remove ~10% of the gastrocnemius-soleus complex muscle mass from 14-16 week old C57BL6 mice. LM-111 enriched fibrin hydrogels were implanted into the VML defect (n=4) and animals were allowed to recover for 2 and 4 weeks. Untreated VML muscles (n=4) and a sham group (n=4) were used as controls.

The hydrogel-treated VML injured muscles showed significant recovery in muscle weight (p=0.0019) compared to the untreated controls. Immunohistochemistry results showed that at 2 weeks post-injury the fibrin and LM-111 hydrogels underwent constructive remodeling at the defect site and supported higher accumulation of satellite cells (Pax7+), hematopoietic cells (CD45+), endothelial cells (CD31+), and macrophages (F4/80+) at the site of injury. Significantly higher protein expression of MyoD (p=0.0053) and Myogenin (p=0.0169) was observed in the hydrogel-treated groups compared to untreated controls. In support, the total number of myofibers with centrally located nuclei also trended higher in the hydrogel-treated VML injured muscles suggesting an increase in myofiber regeneration. Ongoing studies include isometric peak force measurement as well as histological and biomolecular analysis of muscles harvested at 4 weeks post-injury.

30. Ana Maslesa

University of Georgia Regenerative Bioscience Center (1); University of Georgia College of Engineering (2)

Mesenchymal stems cells (MSCs) are a promising therapy for bone diseases, however identification of potent MSCs with high osteogenic potential remains to be addressed. The largest obstacles in MSC therapy include the need for an assay capable of predicting how well a potential MSC therapeutic product will perform under osteogenic conditions. Traditional techniques to measure osteogenic potency include analysis of mineralization, alkaline phosphatase (ALP) production, and gene expression. However, these metrics are only useful in late-stage differentiation. To improve the prediction of osteogenic potency of donor MSCs, we developed an in vitro assay to monitor collagen production throughout the osteogenic process. Collagen is an integral part of bone health, playing roles in both the mechanical and physiological function of bone. Collagen alignment is a novel method of measuring early osteogenic differentiation, with more aligned fibers showing higher osteogenic potency. Here, we imaged donor cell lines with low and high osteogenic potential as measured by mineral production after differentiation. To improve their potency and reduce donor variability, we treated differentiating cells with coumestrol, a phytoestrogen known to increase osteogenic potential of MSCs. In this work, temporal collagen production and secretion was studied using second harmonic generation (SHG) imaging and related to mineralization of the extracellular matrix. In addition, the ALP activity and the morphology of osteo-induced cells were measured and related to collagen formation. It is expected for osteo-inducing compounds such as coumestrol to lessen differences among donors and improve collagen production by MSCs. In future work, we will treat murine MSCs with coumestrol and transplant augmented cells into a metabolic bone disease model and improvement of bone health will be quantified.

Osteoarthritis (OA) is a chronic disease of the joints that leads to degeneration of articular cartilage surfaces. Mesenchymal stem cells (MSC) present a promising treatment to target the disease, relying on their regenerative capacity along with their immunomodulatory and anti-inflammatory properties. However, many questions remain as to the mechanism of action of these cells following intra-articular delivery: paracrine action versus cellular engraftment. Cellular encapsulation presents a promising means to study the paracrine factors of MSCs, independent of cellular engraftment, as the alginate microencapsulation provides a mechanical barrier between the encapsulated cells and the native host tissue. The objective of this study was to quantitatively assess the efficacy of encapsulated hMSCs as a disease modifying therapeutic for OA. OA was surgically induced in rats via the medial meniscus transection (MMT) surgery. The efficacy of hMSC intervention was assessed using Lewis Rats with MMT (n=7-8 per group). Intra-articular injections of hMSCs, or controls (saline, empty capsules or non-encapsulated hMSCs), were given 1-day post-op and animals were euthanized at 3 weeks. Micro-structural changes in the articular cartilage were assessed using equilibrium partitioning of an ionic contrast agent based micro-computed tomography (EPIC-ɥCT). We hypothesized that encapsulated hMSCs would have a therapeutic effect, via paracrine mediated action, on OA progression. Quantitative analysis of articular cartilage, via EPIC-ɥCT, showed attenuated increases in cartilage thickness and surface roughness for the encapsulated hMSC condition in comparison to all other groups. Osteophyte volumes, defined as thickening and partial mineralization of cartilaginous tissue at the marginal edge of joints, were augmented in the encapsulated hMSC group in comparison to all other MMT conditions, except non-encapsulated hMSCs. Furthermore, encapsulated hMSCs yielded increased mineralized osteophyte volumes compared to all other groups. This is the first study to demonstrate that the paracrine signaling properties of hMSCs, independent of direct cellular engraftment, can exert a chondroprotective role in OA. However, these protective effects were countered by enhancements of osteophyte volumes. These augmented tissue volumes are especially relevant in clinical application as many clinical trials are currently ongoing and these findings have yet to be reported.

32. Kuwabo Mubyana

The Influence of Applied Cyclic Uniaxial Strain on the Tensile Properties and Nuclear Shape of Engineered Scaffold-free Tendon Fibers

Kuwabo Mubyana; David T. Corr

Biomedical Engineering Department, Rensselaer Polytechnic Institute

Treating injured tendon is an ever-increasing clinical and financial burden world-wide. Further contributing to this cost is the difficulty restoring full function after injury. Mature tendon is poorly vascularized and, consequently, possesses a poor intrinsic healing capacity. Mechanically inferior scar tissue forms during wound healing, resulting in tendon that is prone to re-rupture. Scaffold-free engineered tendon replacements may provide a promising solution to these challenges.

Recently, our laboratory developed a scaffold-free technique to engineer individual tendon-like fibers in differentially-adherent growth channels, and we utilize this model to study the influence of chemical and mechanical stimuli on fiber structural and functional development. This technique was inspired by key aspects of embryonic tendon development, which include high initial cell density, early application of mechanical stimulation, and fiber formation by cellular self-assembly. The purpose of this study was to examine the influence of applied intermittent cyclic uniaxial strain on the tensile properties and nuclear shape of engineered scaffold-free tendon fibers.

Peak stress, modulus, and toughness all increased with the application of loading and the loading duration. However, toe-in strain and failure strain were uninfluenced by loading. Nuclear area was preserved across groups, but nuclear aspect ratio increased markedly after 3 days of loading. Nuclear shape was of interest because recent studies show that nuclear deformation correlates with altered gene expression, which may give mechanistic insight into the enhanced tensile properties. Our ongoing studies have quantified Col I and Col III intensity and alignment, and will utilize rt-PCR to examine load-induced changes in gene expression of tendon-specific markers.

A limitation with tissue engineering a heart valve is the inability to replicate the trilayer leaflet structure, which is comprised of three unique layers: fibrosa, spongiosa, and ventricularis. The mechanical strength of valves is attributed to the unique microstructure of each valve leaflet layer. Thus, this project aims to develop suitable bioinks for 3D bioprinting a multi-layer scaffold that mimics the microstructure and mechanical properties of the valve leaflets. To generate a multi-layer valve leaflet, a bioink, suitable for 3D bioprinting and cell encapsulation, composed of poly(ethylene glycol) diacrylate (PEGDA) and gelatin methacrylate (GelMA) was developed. Meanwhile, polycaprolactone (PCL) was optimized for 3D bioprinting and utilized as the load-bearing layer of the valve leaflet. Different weight percentages of PEGDA and GelMA were used to determine a bioink that exhibited viscous properties for extrusion-based printing. Bioink properties were characterized by degradation, swelling ratio, and compression test. Cell viability was performed using the Live/Dead Assay. The printability of each bioink was determined by performing a shape fidelity test. All 3D bioprinting was performed using EnvisionTec Bioplotter. A bioink formulation of 10% w/v of GelMA and a combination of GelMA/PEGDA led to a white-light crosslinkable bioink suitable for 3D bioprinting. As for the scaffold stability, the 10% w/v GelMA hydrogels demonstrated complete degradation after 5 days. Meanwhile, GelMA/PEGDA experienced no degradation at Day 7. The volume swelling ratio of GelMa/PEGDA was statistically higher than the 10% w/v GelMA or 10% w/v PEGDA. The mechanical properties of GelMA discs were significantly higher with a modulus of 30 kPa compared to 3D printed GelMA at a modulus of 5 kPa. Furthermore, 10% w/v GelMA and the GelMA/PEGDA demonstrated an average cell viability above 80% at Day 5. The shape fidelity for GelMA/PEGDA was 70% accurate. Meanwhile, the print accuracy for PCL scaffolds were 80% and exhibited a modulus of 4-6 MPa. In summary, this study addresses the need to develop a suitable bioink for 3D bioprinting valve leaflets using GelMA/PEGDA and PCL. Future studies involving the combination of PCL and GelMA/PEGDA are necessary to assemble a 3D printed construct that mimics the microstructure and anisotropic properties of the valve leaflets.

34. Anna Nichenko

Department of Kinesiology, University of Georgia (1); Regenerative Bioscience Center, University of Georgia (2)

Autophagy is a highly conserved cellular process for the degradation of dysfunctional or damaged organelles (e.g., mitochondria). We have previously demonstrated that traumatic muscle injury reduces mitochondrial content and induces autophagy. The objective of this study was two-fold: 1) define the muscle injury-dependent mitochondrial dysfunction and autophagy induction and 2) determine if autophagy is necessary for functional recovery of muscle contractile and metabolic function. To determine muscle injury-dependent mitochondrial dysfunction and autophagy induction, ten week old C57Bl/6 mice were randomly assigned to one of the following groups: eccentric contraction-induced injury (physiological), freeze injury (traumatic), or contractile fatigue (non-damaging control). Injured and contralateral uninjured tibialis anterior muscles were harvested at 0, 6 hours, 1, 3, and 7 days. Mitochondrial function was assessed via state 3 mitochondrial respiration rates from permeabilized muscle fiber bundles, and autophagy induction was measured via Beclin1, LC3-II, and Ulk1 protein content. Only freeze injury resulted in a decline in mitochondrial function (p<0.01), whereas both eccentric contraction-induced injury and freeze injury resulted in a robust autophagy response (p=0.02 and p<0.01, respectively). This supports a muscle injury-dependent hypothesis, yet suggests a different physiological threshold exists between mitochondrial dysfunction and autophagy induction. To determine if an autophagy response is necessary for the recovery of mitochondrial function, we subjected skeletal muscle-specific Ulk1 knockouts (Ulk1 KO) and their littermate controls (LM) to freeze injury and assessed the recovery of mitochondrial function and muscle strength after 14 days. Ulk1 KO mice had less recovery of muscle strength (p<0.01), but there was no difference in the recovery of mitochondrial function between KO and LM mice. Additionally, there was no significant difference in Ulk1 protein expression in the injured limb between KOs and LMs. The difference between contractile and mitochondrial functional recovery in KO mice may be explained by the partial rescue of Ulk1 expression in injured KO limbs (potentially by satellite cell-derived Ulk1), a disconnect between O2 consumption and ATP production in injured limbs, or divergent signaling pathways for the interaction of Ulk1 in the recovery of muscle strength and mitochondrial function. In conclusion, autophagy is induced following both traumatic and physiological muscle injury and Ulk-1 mediated autophagy may be required for the recovery of muscle strength. Further studies should be performed to decipher the difference between contractile and mitochondrial functional recovery in Ulk1 KO mice.

35. Alexis Nolfi

McGowan Institute for Regenerative Medicine (1); Department of Bioengineering, University of Pittsburgh (2); Department of Obstetrics, Gynecology, and Reproductive Sciences, University of Pittsburgh (3)

Polypropylene mesh is commonly used for the repair of tissue in pelvic organ prolapse and stress urinary incontinence, as well as for hernia repair. Unfortunately, polypropylene mesh is associated with complications including mesh exposure through tissues, pain, and infection. In excised tissue from patients experiencing a complication, the mesh-tissue-interface is characterized by an abundance of macrophages exhibiting a pro-inflammatory phenotype. Macrophages, however, are plastic cell types with phenotypes along a spectrum of pro-inflammatory and pro-remodeling/anti-inflammatory extremes. It has been demonstrated that modulation of macrophage phenotype during initial stages of healing could prevent chronic inflammation, improving downstream outcomes. Previous work has shown that an IL-4 eluting coating for polypropylene mesh initially polarizes macrophages to the pro-remodeling/anti-inflammatory phenotype, which results in a mitigation of the foreign body reaction downstream. However, timing and duration of the in-vivo immunomodulatory release of IL-4 (and its effect on macrophage phenotype transition) is important for timely shift to anti-inflammatory phenotypes and eventual resolution of inflammation. To customize the release of IL-4 in a spatial and temporal way, we aim to use fluorescently tagged IL-4 with live-animal in-vivo imaging in animals implanted with coated mesh. Successful visualization of IL-4 release will be important for eventual varying of release profiles in order to correlate coating patterns to downstream integration outcomes.

IL-4 was subjected to fluorescent labeling with Alexa Fluor 594 and assessed with HPLC. In order to provide the most physiologically relevant in-vivo release profile, it is important that the fluorescently tagged protein maintain bioactivity. IL-4 polarizes macrophages to a pro-remodeling phenotype with increased arginase-1 production; therefore, tagged IL-4 vs untagged IL-4 was supplemented into the media of naïve macrophages. The in-vitro culture assay showed that tagged and untagged IL-4 produced equivalent levels of increased arginase-1 when compared to macrophages that were cultured in media without supplementation, showing that the fluorescent tag does not affect the bioactivity. Finally, tagged IL-4 was loaded into a dermatan sulfate-chitosan electrostatic layer-by-layer coating of polypropylene mesh using previously established protocols and imaged. Confocal imaging showed a uniform signal in the red channel for mesh that is coated with fluorescently tagged IL-4, indicating that the tagged protein is able to be incorporated into the dermatan sulfate-chitosan coating. In-vivo implantation of this tagged mesh will allow daily live-animal imaging of the same animal until loss of signal so that release profiles can be manipulated and then correlated to downstream outcomes.

36. Austin Passaro

Regenerative Bioscience Center, University of Georgia (1); Division of Neuroscience, University of Georgia (2); Biomedical Health and Sciences Institute, University of Georgia (3); Department of Mechanical Science and Engineering, University of Illinois at Urbana-Champaign (4); Department of Animal and Dairy Science, University of Georgia (5)

Modeling the neuromuscular junction (NMJ) remains an important goal to explore regenerative therapies for neurodegenerative disorders and injury. Most current in vitro NMJ models involve simple co-cultures of motor neurons or spinal cord explants and muscle cells and are validated by overlap of neuronal endplates and acetylcholine receptors (AChR) via immunocytochemistry. If functional validation is performed at all, it is typically via patch clamp or calcium imaging of individual neurons. While these models are useful, they are not particularly physiologically relevant for a few reasons: simple co-cultures do not recapitulate spatial separation of neurons and muscle cells, simple co-cultures generally lack considerable three-dimensionality, glia are often absent, and functional studies are difficult to perform due to the lack of organization and inability to precisely stimulate neurons.

Using optogenetically active EBs, the motor neurons can be stimulated, producing a contractile response suggestive of functional NMJ formation. This response is blocked by curare treatment (an AChR inhibitor), providing further evidence that the observed contraction is a result of a functional NMJ.

Finally, we used a multielectrode array (MEA) system to analyze neural activity of the EBs seeded into the platform to validate that these cells spontaneously form neural networks. Interestingly, we not only demonstrate that the cells have considerable spontaneous activity, but also that they display significantly increased network activity in the presence of myotube-conditioned media.

Taken together, these results provide evidence that this platform allows for functional NMJ formation. Additionally, we posit that this platform has distinct advantages and is more physiologically relevant than traditional co-cultures due to its compartmentalization and three-dimensionality, making it valuable for the study of neurodegenerative diseases and regenerative therapies, as well as for drug discovery and tissue engineering applications.

University of Georgia Regenerative Bioscience Center (1); University of Georgia College of Engineering (2)

Mesenchymal stem cell (MSC) therapy has promise to treat a multitude of diseases and is currently in more than 300 clinical trials world-wide, but their promise has yet to translate into therapy. Inability to identify potent cells for therapy and assess therapeutic endpoints are major contributors to failures in MSC therapy. Current assays use mineralization, gene expression, and alkaline phosphatase (ALP) production to describe MSC potency for the treatment of bone disease, which are predominately expressed late in the differentiation process. One underexplored potential early and definitive marker for osteogenic differentiation is collagen type 1, which serves as the bone matrix framework for mineral deposition. Thus, we seek to develop a method to characterize collagen using a non-destructive label-free imaging strategy that could be integrated into a cell manufacturing workflow and enable prediction of MSC osteogenic outcomes. To do so, we evaluate second harmonic generation (SHG) signal produced by collagen fibers using our home-built two-photon microscope. Using this technique, we can describe collagen formation and secretion during early stages of MSC differentiation. In this work, we evaluate the rate and quantity of collagen production, and use polarization resolved SHG imaging to quantify the orientation of secreted collagen fiber bundles. Our collagen production quantification technique in high and low osteogenic potency MSC donors closely follows traditional terminal strategies for assessing MSC osteogenic production, including assessment of mineralization, gene expression, and ALP production. This label-free and non-destructive imaging assay identifies cells with high in vitro osteogenic potency early in the MSC production process. We anticipate the in vitro potency will be predictive enhanced in vivo bone healing results. Future work will leverage SHG imaging to study potential methods for increasing collagen production, and will seek to understand the optimal state of MSC differentiation for transplantation into the bone.

McGowan Institute for Regenerative Medicine (1); Department of Bioengineering, University of Pittsburgh (2); Department of Surgery, University of Pittsburgh (3); School of Medicine, University of Pittsburgh (4)

The molecule 4-hydroxybutyrate (4HB) is a naturally occurring bioactive endogenous short chain fatty acid (SCFA) that has been used for the production of polymeric mesh materials for soft tissue repair applications. The 4HB monomer has been exhaustively studied for its role as metabolite and modulator of the neurotransmitter g-aminobutyric acid (GABA) in the central nervous system (CNS). Other functions of 4HB within non-CNS tissues have been less studied; however, this SCFA is a hydroxylated form of butyrate, a known histone deacetylase (HDAC) inhibitor secreted by commensal bacteria within the gastrointestinal tract. Butyrate reportedly exerts its immunomodulatory functions by suppressing pro-inflammatory macrophages and promoting antimicrobial peptide (AMP) secretion. However, the immunomodulatory effects of 4HB upon cells of the immune system and the ability of 4HB to induce the expression of AMP have not been studied. The present study evaluated the effect of 4HB upon macrophage phenotype and the expression of AMP, and the molecular mechanisms by which such effects are mediated.

The monomer 4HB was used in-vitro to evaluate the activation of markers associated with the pro-inflammatory (iNOS+) and anti-inflammatory (Fizz-1+, Arginase-1+) phenotype of murine bone marrow-derived macrophages. The expression of the AMP cathelicidin LL-37 and β-defensins were also determined. The molecular mechanisms involved in the expression of AMP were evaluated by gene expression and inhibition immunoassays to target intermediate proteins within the pathway. In addition, a surgical mesh material composed of poly (4-hydroxybutyrate) (P4HB) was evaluated in-vivo using a rat bilateral partial thickness abdominal wall defect model. Immunolabeling quantification was used to determine the immunomodulatory effects of P4HB upon macrophage phenotype, using CD-68+/CD206+ (anti-inflammatory), and CD-68+/CD-86+ (pro-inflammatory) markers, and cathelicidin LL-37 expression, at 3, 7, 14, 21, and 35 days post-implantation.

The results show the ability of 4HB to promote an anti-inflammatory, regulatory macrophage phenotype and increased expression of AMP. The associated molecular mechanisms involve transcriptional activation of the genes codifying the AMP through the MAP-kinase pathway. The results expand the understanding of the biologic activity of 4HB in cells of the immune system, and its potential to promote a constructive modulatory effect for regenerative medicine applications.

39. Robby Ratajczak

Modulating MSC Binding via Nitric Oxide-based Treatment

Robby Ratajczak; Bobby Leitmann; Hitesh Handa; Luke J. Mortensen

Driftmier College of Engineering, University of Georgia

Mesenchymal stem cells (MSCs) have shown to have great potential for cell based therapies due to their natural anti-inflammatory aspects and ability to help rehabilitate damaged or distressed tissues. MSCs demonstrate poor homing to inflamed sites when introduced to the body systemically. Without proper homing, their potential as a cell based therapy is lessened. Through the process of small molecule treatment, MSCs can be engineered to modulate different adhesion molecules which will allow for better homing. Treatment of MSCs with nitric oxide is hypothesized to reduce the cellular expression of vascular cellular adhesion molecule 1 (VCAM1), which we hypothesize will reduce the susceptibility for cells to bind to off-target sites or get stuck in filtering organs on the way to the desired site of inflammation. Nitric Oxide has also been shown to increase the lifespan of MSCs, which will aid in their ability to reach the site. In this project we will use the nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP) to reduce cellular adhesion. MSCs will be pretreated with SNAP to expose the cells to nitric oxide and then studied to assess its effect on their homing. Previous studies have shown that nitric oxide does affect the adhesion of cells when treated in suspension with more than .5 mM of SNAP. Nitric oxide cytotoxicity was analyzed through in vitro viability assays (alamar blue) and run through flow adhesion assays to test their adhesion to an endothelial monolayer under physiological conditions. Furthermore, VCAM1 expression was found to be reduced using antibody binding and qPCR after nitric oxide treatment. We hypothesize that nitric oxide treatment will significantly increase homing due to reduced tendency to bind to off-target sites. If our data proves this hypothesis to be true, MSC potential for applications in cell based therapies for inflammatory or immune conditions such as Canine Atopic Dermatitis will increase significantly.

40. Cheryl San Emeterio

In recent years, human proteins extracted from hair clippings (an unregulated waste material) have become recognized as having biological activity important for wound healing.1 We previously exploited the reversible, molecular self-assembly attribute of keratins and keratin-associated proteins to make a clear matrix that is purely comprised of reconstituted human proteins without the use of additives or chemical crosslinking agents2, now branded as ProgenaMatrix™, and demonstrated wound-healing efficacy on par with allograft human tissues.3

The immune response that is rapidly initiated after skin injury follows a precise sequence from the acute inflammatory phase, to the later anti-inflammatory phase. Failure to progress to the anti-inflammatory, or repair phase results in chronic wounds. Within the later anti-inflammatory phase of healing, pro-regenerative “M2” macrophages promote and coordinate a wide range of processes including angiogenesis, extracellular matrix deposition and remodeling, and inflammation resolution. Though M2 macrophages likely exist in a wide range of phenotypes in vivo, certain surface markers have been shown to broadly define macrophage populations that exhibit pro-regenerative functions within injury contexts. Notably, CD206 is a widely used marker for M2 macrophages, whereas CD301b (dually expressed with CD206) has been more recently shown to mark a population of pro-regenerative macrophages specifically involved in dermal repair. Based on this knowledge, the innate inflammatory response to ProgenaMatrix™ was evaluated in a full-thickness excisional skin injury model in both C57bl/6 and db/db mice.

We first evaluated the innate inflammatory response to ProgenaMatrix in wildtype C57bl/6 mice. Briefly, two 6mm full thickness wounds were created on the dorsum of each mouse, and one wound left untreated, and one wound treated with ProgenaMatrix. Interestingly, we observed that at day 2 post-surgery, ProgenaMatrix induces a significantly higher number of live cells within wound beds as well as higher viability. Tissue weights of the biopsy punches were not significantly different between groups. This higher live cellularity within ProgenaMatrix treated wounds manifests in several compartments of the innate immune response to skin injury. CD11b+ myeloid cells, neutrophils, and non-classical, anti-inflammatory monocytes are significantly increased with ProgenaMatrix treatment. Interestingly, classical, inflammatory monocytes are not significantly different between groups at day 2. Notably, pro-regenerative F4/80+ macrophage subtypes (CD206+Ly6C-, and CD206+CD301b+ macrophages) are significantly increased with ProgenaMatrix treatment. CD206+CD301b+ macrophages have been shown to be required for full-thickness skin wound repair and govern fibroblast proliferation and repopulation of wound beds. When we performed the same flow cytometry analysis on cells within day 5 wound beds, there was no difference between untreated and ProgenaMatrix in any analyzed cell population. These results indicate that ProgenaMatrix acts acutely within healthy mice to enrich wounds with pro-regenerative macrophages that can then coordinate favorable healing outcome.

We then compared the accumulation of immune cells at 5 days post-surgery between wild-type C57bl/6 mice and delayed-healing db/db mice. Interestingly, diabetic mice exhibit significantly lower numbers of live cells both in untreated and ProgenaMatrix treated wounds (Fig 6a). However, though there was a significant decrease in viability of cells within untreated wounds in diabetic mice compared to wildtype, this decrease was not observed when comparing ProgenaMatrix treated wounds between wildtype and diabetic animals. This “rescue” of diabetes-associated decrease in viability is mirrored when looking at the numbers of CD11b+ myeloid cells, F4/80+ macrophages, and CD206-Ly6C+ inflammatory macrophages. Notably, ProgenaMatrix not only rescued the diabetes-associated decrease in CD206+Ly6C- macrophages and CD206+CD301b+ macrophages in day 5 wounds, but also increased their number relative to untreated diabetic wounds. Taken together, these findings shed light on the immunological mechanism behind diabetes-associated delayed wound healing, and indicate that the efficacy of ProgenaMatrix lies in its ability to promote cell viability within chronic wounds and enrich the wound environment with pro-regenerative immune cells that facilitate accelerated tissue repair.

Department of Bioengineering, School of Engineering, University of Pittsburgh (1); Department of Plastic Surgery School of Medicine, University of Pittsburgh (2); Department Biology, School of Arts and Sciences, University of Pittsburgh (3); Department of Chemical Engineering, School of Engineering, University of Pittsburgh (4); Department of Biomedical Engineering and Orthopaedic Surgery, University of Virginia (5)

Atrophic dysfunctional muscle resulting from peripheral nerve injury poses an exceptional, and to date, unmet challenge for regenerative medicine. Nearly $150 billion in healthcare is spent annually on peripheral nerve injury and the resulting muscular atrophy after denervation. Successful regrowth of the injured nerve does not necessarily lead to restoration of muscle power and function, especially if combinative therapies addressing both the nerve injury and the muscle atrophy are not used. Here, a rodent model for muscle atrophy of the gastrocnemius results from a 1.5cm defect to the sciatic nerve. To address the nerve injury, autograft was placed into the defect representing the standard of care (positive control, n=4). Additionally, allogeneic rodent adipose-derived stem cells were injected into the gastrocnemius post-operatively in two cohorts, one receiving a single injection (n=5), and the other receiving two injections, post-operatively and at three weeks (n=5). A fourth cohort received neither autograft nerve nor ASC therapy after creation of the nerve defect (negative control, n=3). At six weeks, the cohorts having received an ASCs injection post-operatively had a higher muscle mass percentage retained, being 27% upon normalization to the non-operated gastrocnemius, compared to the negative control retaining only 14% muscle mass (p=0.000). The single injection of ASCs resulted in greater average fiber area, 861 mm2 compared to 683 mm2 in the standard-of-care cohort but was significantly less than the average fiber area pooled from the non-operated muscle across conditions, quantified as 2917 mm2 (p=0.000). Further, a single injection of ASCs in conjunction with nerve autografting was shown to have less overall lipid content accumulated throughout the musculature compared to standard of care. Additionally, muscles having received ASCs injection showed increased presence of IL-10 and Ki67, with decreased presence of iNOS. Collectively this investigation is suggestive that an ASCs injection into denervated muscle post-operatively is able to delay the onset of atrophy.

Division of Cardiology, Emory University School of Medicine (1); Department of Otolaryngology, University of Arizona College of Medicine (2); Department of Cardiology, Xiangya Hospital of Central South University (3); Department of Chemistry, Emory University (4); Woodruff School of Mechanical Engineering and Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology (5); Department of Pediatrics, Emory University School of Medicine (6);

Myocardial ischemia/reperfusion (MI/R) injury releases immunogenic metabolites that stimulate innate immune cells to infiltrate vulnerable myocardium and drive tissue devitalization. Mesenchymal stromal cells (MSCs) are a potential treatment modality for MI/R due to their anti-inflammatory capabilities; however, the manner in which they regulate the acute inflammatory milieu requires further elucidation. Activity of the ecto-5’-nucleotidase, CD73, may play a critical role in inflammatory regulation by converting pro-inflammatory adenosine monophosphate (AMP) to anti-inflammatory adenosine (ADO). The purpose of this study is to determine whether MSC-mediated conversion of AMP to ADO reduces inflammation in early MI/R injury, favoring a micro-environment that attenuates excessive immunity and facilitates earlier cardiac recovery.

Adult rats were subjected to 30 minutes of MI/R injury. MSCs were encapsulated (eMSCs) with alginate hydrogel and implanted on the myocardial surface with VPM-crosslinked PEG at the time of reperfusion. A subset of MSCs were pre-treated CD73 inhibitor, α,β-methylene adenosine diphosphate (APCP), prior to implantation. Saline or hydrogel vehicle were used as controls. One day following injury we measured myocardial ADO content by mass spectrometry, hydrogen peroxide (H2O2) formation by Amplex Red assay, and leukocyte infiltration by flow cytometry. Cardiac function was measured by speckle-tracking echocardiography for 28 days following MI/R. eMSC capsules were explanted at 1 and 3 days post MI/R to determine MSC viability.

Our study identifies a novel mechanism by which MSCs regulate excessive inflammation by increasing local ADO bioavailability through CD73 enzymatic function when implanted following MI/R injury. This work adds to the growing mechanistic understanding of how cell-based therapy is beneficial in cardiovascular disease states, and may assist is establishing optimal use of such therapies in further pre-clinical research. Future studies will utilize CD73-deficient MSCs to further determine its role in the regulation of innate immunity in MI/R.

Glaucoma is the second most common cause of blindness, and is frequently associated with elevated intraocular pressure, which is largely controlled by a specialized tissue known as the trabecular meshwork (TM). TM cellularity is reduced in glaucoma, and thus the use of stem cells to restore proper TM cellularity and function is of great interest as a glaucoma therapy. To realize this goal, delivery and engraftment of stem cell into the TM is needed. To enhance delivery, we here use magnetic nanoparticles to label mesenchymal stem cells (MSCs) and magnetically pull them to the TM.

Adipose-derived MSCs (Lonza) were labeled with either small (20nm) or large (200nm) Prussian blue iron oxide nanocubes (PBNCs) and fluorescently tagged with CFSE for histology. MSCs were injected (250,000-1 million cells/eye) into porcine eyes maintained in organ culture while hydrostatically clamped at physiological pressure levels. Neodymium magnets, arranged in a ring or single “bar” format, were placed near the limbal region of eyes to pull MSCs to the TM. Next day, eyes were fixed and en face images of the TM circumference were captured by fluorescence microscopy. We also developed a model of glaucoma based on TM cell loss induced by graded levels of H2O2-induced oxidative stress in perfused porcine eyes, and delivered cells into these eyes.

Overnight incubation with PBNCs resulted in MSCs being successfully labeled. After MSC injection into eyes, 200nm diameter PBNCs resulted in preferential cell delivery to the TM quadrant adjacent to a permanent bar magnet. In the case of 20nm PBNCs, the benefit was modest. However, when using 200nm PBNCs, 15-fold more cells were delivered to the TM vs unlabeled MSC controls; further, “steering” was rapid, occurring in less than 1 hour. A ring magnet enhanced the circumferential uniformity of MSC delivery to the TM. Finally, MSC uptake into the TM was facilitated in partially decellularized eyes, presumably due to increased availability of cell binding sites.

We conclude that MSC steering to the TM is feasible with the use of PBNCs. Further optimization of magnet design is needed to minimize off-target delivery of MSCs to other locations in the anterior eye. Fortunately, PBNC nanoparticles are optical absorbers and can also be used in ultrasound/photoacoustic imaging to track MSC delivery in real time and longitudinally.

Ongoing advances in transplantation surgical techniques, together with improvements in immunosuppression regimens (IS), have led to a reduction in the rate of postoperative morbidity and graft loss1. Despite these advances, wound complications due to systemic IS are a common post-transplant surgical complication, and systemic IS regimens are associated with an increased risk of tumor formation, opportunistic infection, and organ toxicity2. Therefore, there is a need for therapies that have the ability to allow for wound healing while also inducing transplant tolerance without the off-target side effects of systemic IS. One method that could mediate these complications is the delivery of immunomodulatory factors that are able to induce transplant tolerance through the local recruitment and education of tolerogenic subsets of immune cells that work to enhance alloimmunity by dampening immune activation and cytokine production of factors involved in rejection.

We engineered an enzymatically degradable poly(ethylene) glycol (PEG)-based hydrogel system for the controlled delivery of interleukin 10 (IL-10). IL-10 is necessary for T-regulatory (Treg)-mediated allotolerance and can participate in the local education of DCs capable of promoting Treg activity and allograft acceptance. We investigated the modulation of myeloid cell recruitment and intracellular cytokine expression to assess differentiation of tolerogenic cell phenotypes, while also evaluating longitudinal peri-implant vascular changes.

Delivery of IL-10 from subcutaneously implanted hydrogels was able to modulate myeloid cell recruitment to peri-implant tissue as both days 3 and 7 after implantation. At day 3, the presence of Ly6CLo Anti-inflammatory monocytes (AMs) was significantly increased near IL-10-loaded hydrogels compared to unloaded control. Likewise, at seven days, there were significantly more CD206+ M2 macrophages present around the IL-10-loaded gels. Intracellular staining revealed significantly reduced TNF-a staining and significantly increased IL-10 staining in CD11c+ dendritic cells. Intravital brightfield imaging of vasculature in the IL-10 peri-implant space reveals changes in the tortuosity and vascular diameter not seen after implantation of vehicle.

45. William Southern

Department of Kinesiology, University of Georgia (1); Regenerative Bioscience Center, University of Georgia (2)

Volumetric muscle loss (VML) is characterized by a large volume of muscle tissue being removed from the body due to surgery or severe trauma. Currently, clinical rehabilitation approaches following VML are poorly defined and have limited efficacy. The objective of this study was to evaluate the efficacy of early onset rehabilitative modalities to improve muscle function in the remaining muscle tissue following VML. VML injury was performed unilaterally on the gastrocnemius and soleus muscles of 8-week old C57BL/6 mice. Rehabilitative interventions began 72-hours after injury. Mice were divided into one of the following groups (n=4-7/group) for 4 weeks: no therapy (VML alone), electrical stimulation training eliciting concentric (i.e., shortening) contractions (VML+CON), electrical stimulation training eliciting eccentric (i.e., lengthening) contractions (VML+ECC), or voluntary wheel running (VML+WR). Muscle mass, peak isometric torque, passive muscle stiffness, and muscle collagen content of the plantar flexor muscles were assessed in all groups. Rehabilitation had no effect on the recovery of injured gastrocnemius mass (VML alone vs. any of the rehabilitation groups, P=0.68). Peak isometric torque of the plantar flexor muscle was ~46% greater in VML+CON and VML+WR mice compared to VML alone (P=0.025) suggesting that CON and WR interventions beneficially remodeled the remaining muscle tissue, independent of muscle mass. Passive muscle stiffness in the injured limb, which is an important metric of joint mobility, was not different among VML alone, VML+ECC, and VML+CON groups, but was 68% greater in VML+WR (P<0.001). Interestingly, the higher muscle stiffness in the VML+WR mice was not accompanied by greater collagen content. In fact, the injured limb of VML+WR mice had the lowest collagen content of any groups (P=0.016) suggesting that wheel running alters collagen orientation and/or type. In conclusion, early initiation of low-force generating rehabilitative interventions (i.e., concentric contractions and WR) are able to positively alter muscle function by potentially remodeling muscle architecture of the remaining muscle. Therefore, early onset of low intensity, multi-muscle, weight bearing exercises may be a valuable clinical approach for persons with VML.

46. Benjamin Spearman

J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida

Hyaluronic acid (HA) is an ubiquitous extracellular matrix (ECM) component commonly used in soft tissue engineering. HA can be modified in many ways including via methacrylation to tune the mechanical properties and allow for UV crosslinking. Two methods of methacrylation were used including via glycidyl methacrylate and methacrylic anhydride resulting in GMHA and MAHA, respectively. These components were also combined with other ECM components, including collagen I and laminin (to provide cell adhesive properties to the hydrogels). Methacrylation of GMHA and MAHA was confirmed and degree of methacrylation quantified with HNMR. The hydrogels were mechanically characterized by indentation. Increased concentration resulted in increased modulus. For example, GMHA’s modulus increased from 1.8 to 4.0 to 4.8 kPa at concentrations of 5, 10, and 15 mg/mL, respectively; while MAHA’s modulus increased from 5.1 to 6.1 kPa at concentrations of 5 and 10 mg/mL. This is in the same order of magnitude as rat sciatic nerve which had a modulus of 2.5 kPa. Both GMHA and MAHA hydrogels with collagen and laminin were not significantly different from rat sciatic nerve. Schwann cells were cultured on 6 different hydrogels, including GMHA and MAHA at concentrations of 5 mg/mL, triple component gels with either GMHA or MAHA plus collagen I and laminin, collagen I alone, and collagen I plus laminin. An Alamar blue assay was performed 1, 3, and 7 days following Schwann cell plating on hydrogels to assess cell viability. Cell viability was normalized to Day 1 on collagen alone. Both GMHA and MAHA saw decreases in cell viability from Day 1 to Day 3. However, the addition of collagen and laminin resulted in continued increase in cell viability from Day 1 to 3 to 7. In conclusion, a variety of hydrogels were designed using two different methacrylated HA with the addition of collagen I and laminin. A range of mechanical properties were obtained by modifying the concentration of methacrylated HA in solution or adding other ECM components and were similar to that of native rat nerve. Schwann cell viability was found to increase by adding collagen I and laminin to the methacrylated HA. This work will provide a platform to develop an assortment of tunable ECM-based hydrogels for a variety of soft tissue engineering applications. Future work will focus on additional tunability of the hydrogels by modifying the degree of methacrylation of MAHA and GMHA.

47. Samantha Spellicy

Multiparametric Analysis of Structural and Functional Correlations in a Porcine MCAO Model of Stroke Reveals Divergent Trends between Treatment Groups over Time

Magnetic resonance imaging (MRI) has become a pivotal tool for diagnosis and management of stroke in clinical medicine due to its increased specificity and sensitivity over other commonly used imaging modalities. More information is still needed, however, to discover how these structural measurements, determined through MRI, correlate to the functional and behavioral deficits of these in animal models of stroke. To help answer these questions we conducted a porcine middle cerebral artery occlusion study (MCAO) study. From this study we wish to demonstrate that our multiple parameter correlation matrices allow for a more holistic understanding of the relationship between structural and functional changes we are observing in our pig MCAO model of stroke, as well as reveal differences between our treatment groups we would have not observed through single parameter analysis.

In this study, we invoked a right sided MCAO in 16 pigs, and divided pigs into treatment or sham group. We administered neural stem cell-derived extracellular vesicles (NSCEVs) for our treatment group and PBS for our sham group at 2, 14, and 24 hours. MR Imaging was conducted at 24 hours following induction of stroke and gait and behavior was then monitored on days 1, 3, 7, 14, 21, 28, 56, and 84 post-stroke. Pigs underwent their second MRI at day 84 and then were sacrificed for histological analysis. During the testing days we collected data on 65 gait and 25 behavior parameters. We then ran pairwise correlations of these parameters against 16 measured structural MRI parameters. These correlations were conducted separately for each group and each day tested.

In this study, we identified a subset of MRI parameters including, T2W left hemisphere volume, T2W right hemisphere volume, T2W lesion volume, and FA internal capsule which were significantly correlated to gait and behavior parameters on day 1 post MCAO. We found 13/90, 20/90, 14/90 and 14/90 functional parameters had significant correlations to these respective MRI measurements in our sham group, compared to only 3/90, 2/90, 4/90, and 2/90 in our treated group. In our treated NSCEV group, axial midline shift had the greatest number of functional correlations at day 1 with 10/90, which was also strongly correlated in untreated group with 18/90. Day 84 gait and behavior correlations to Day 1 MRI parameters revealed divergent trends between the directionality of correlations between the treated and sham group. For example, there was a significant positive correlation in our treated group between T2W left hemisphere and normalized mean pressure on the right paw, but a negative correlation in these same parameters for our sham group.

Overall, we found that we could correlate specific structural parameters measured on MRI post MCAO to functional deficits found in behavior and gait in the pigs. From this multiparameter correlation analysis, we were able to pick out differences between the two treatment groups which were previously undetectable. Overall, this correlation analysis highlighted important clinically relevant MRI measurements which will be important to utilize moving forward because of their impactful correlation to functional changes and effectiveness in discerning between treatment groups.

48. Elizabeth Stahl

Evaluation of the Host Immune Response to Decellularized Lung Scaffolds Derived from Wild Type or -Gal Knockout Pigs in a Non-human Primate Model

McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA (1); Department of Bioengineering, Swanson School of Engineering, University of Pittsburgh, Pittsburgh, PA (2); Cellular and Molecular Pathology Training Program, University of Pittsburgh School of Medicine, Pittsburgh, PA (3)

The development of -Gal negative porcine tissues is an important step to overcome challenges of xenogeneic organ transplantation for human patients, as decellularization strategies alone do not completely remove -Gal epitopes. Humans and Old-World monkeys, such as the rhesus macaque, do not express the -Gal epitope and thus develop antibodies against -Gal, leading to complement activation and hyperacute rejection following exposure to porcine tissues.

In the present study, lungs from wild type or -Gal knockout pigs (GalSafe®) were harvested and decellularized to remove cellular antigens and implanted subcutaneously in a rhesus macaque model (n=8). Native porcine lungs and a sham injury group were included as controls. The scaffolds were explanted at 1, 2, 4, and 8 weeks then fresh scaffolds were re-implanted into sensitized hosts to evaluate adaptive immunity. Histological analyses (H&E, Masson’s Trichrome, and Verhoff-Van Gieson staining) were evaluated qualitatively and quantitatively. Markers of inflammation and wound healing were also examined using immunohistochemistry (CD4, CD8, CD45, CD20, CD68, CD86, CD31, CD1, CD206, and alpha-smooth muscle actin).

The GalSafe® decellularized lung implant performed similarly to the wild type decellularized lung implant in the acute study, and induced more CD3+ T-cells, primarily CD4+ helper subsets, compared to the other implantation groups. However, upon re-implantation into sensitized hosts, the GalSafe® decellularized lung implant had reduced CD3+ T-cell infiltration and no change in CD20+ B-cell levels, likely a result of reduced antibody-mediated immunological memory, while other groups had a more robust adaptive immune response. Overall, the GalSafe® decellularized lung implant performed similarly to the wild type decellularized construct in an acute setting, and shows promise as a potential strategy to overcome chronic immune challenges in xenotransplantation and regenerative medicine applications.

Regenerative Bioscience Center, University of Georgia (1); Department of Chemistry, University of Georgia (2); Department of Electrical and Computer Engineering, University of Georgia (3)

Chondroitin sulfate glycosaminoglycans (CS-GAGs) are important regulators of neuronal homeostasis in the brain extracellular matrix. However, their use as scaffolds for neural tissue engineering has been underexplored. We have previously demonstrated that photopolymerized CS-GAG matrices consisting predominantly of monosulfated GAGspromoted NSC efficacy and enhanced neuroprotection after a severe traumatic brain injury (TBI). In this study, we use copper-free “click” chemistry to fabricate porous CS-GAG matrices capable of maintaining the undifferentiated state of neural stem cells (NSCs), or selectively facilitating neuronal differentiation as desired.

Polyethylene glycol (PEG) amine functionalized dibenzocyclooctynol (DIBO) was covalently coupled to the carboxyl group on the glucuronic acid on the CS-GAG chain at a molar ratio of 0.6:1. Aliphatic azide functionalized PEG is coupled to the CS-GAG chains similarly to yield azido-CS. 2.5% w/v DIBO-CS and 1% w/v azido-CS in basal media were combined to yield porous hydrogels. For neuronal differentiation studies, NSCs were encapsulated in L1 peptide functionalized hydrogels. Azide tagged L1 peptides (Az-PSITWRGDGRDLQEL, 5mM) are reconstituted with azido-CS to yield L1-azido-CS, and cross-linked with DIBO-CS as described above. Hydrogels are allowed to crosslink for minimum of five minutes at room temperature. Reaction products were validated using 1H NMR, and material properties were characterized using SEM, rheology, and swelling and degradation assays. Human induced pluripotent (HIP) NSCs were suspended in cell culture medium and either seeded on top of pre-fabricated hydrogels, or encapsulated within hydrogels. Layered cell-laden hydrogel complexes to mimic the layered architecture of the cerebral cortex were fabricated by dispensing hydrogel solutions containing NSCs, with and without L1 peptide, sequentially into fluorinated ethylene propylene tubes (0.8mm inner diameter). The interaction between differentiated neurons and undifferentiated NSCs encapsulated in layered hydrogel constructs was evaluated after immunohistochemical staining and validation of tri-lineage markers using light sheet fluorescence microscopy (LSFM).

The study demonstrates the potential utility of differentially functionalized and tunable CS-GAG “click” hydrogel matrix layers of defined structural properties to study NSC maintenance and differentiation, and as scaffolds for functional neural tissue repair after a severe TBI.

By virtue of lymphatic drainage mechanisms, pathways of tumor immune suppression have the potential to be active in both the tumor microenvironment as well as sentinel lymph nodes. However, the effects of increased tumor vascular permeability as well as diminished lymphatic function on the capacity of tumor-secreted macromolecular species (e.g. cytokines, exosomes, etc.) active in the regulation of immune signaling to distribute amongst subpopulations of sentinel lymph node-resident cells that exhibit distinct lymph node substructural distributions as well as immunological functions remains unexplored. We therefore sought to evaluate the effects of tumor vascular remodeling, including those resulting from tumor vascular endothelial growth factor (VEGF)-C overexpression (OE) and VEGF receptor-2 inhibition, on exposure of sentinel lymph node-resident immune cells to factors derived from the tumor microenvironment. To accomplish this goal, we analyzed the in vivo biodistribution and uptake by immune cell subtypes of a panel of near-infrared fluorescently labeled macromolecular tracers size-matched to physiological biomolecules ranging from 10-500 nm in hydrodynamic diameter after intradermal or intratumoral infusion in C57Bl6 mice in a temporal- and tissue-resolved fashion. Our results indicate that both VEGF-C OE and VEGFR-2 inhibition partially attenuate tumor vascular leakiness and restore dendritic cell trafficking to sentinel lymph nodes and that VEGFR-2 inhibition but not VEGF-C OE recovers the accumulation of lymph-transported tumor-derived macromolecules within cell subpopulations resident within distinct localities in sentinel lymph nodes in a hydrodynamic size-regulated manner. Our findings have important implications in the role of tumor lymphatic transport in the immunological crosstalk between tumors and their draining lymph nodes, as well as in the development of drug delivery strategies to mitigate its deleterious effects.

Ligament reconstruction is needed when there is a complete tear or rupture of the ligament, resulting in joint instability. Autografts and allografts are commonly used for the surgical repair of ligaments. However, these grafts incur many problems such as donor site morbidity, risk of infection, low cellularity, and limited revascularization. Due to these concerns, ligament tissue engineering (TE) is an active area of research. An electrospun silk fibroin scaffold could be an advantageous scaffold for ligament TE, since silk-based scaffolds are biocompatible, support cell adhesion, and promote proliferation. Silk fibroin also has a hierarchical structure that mimics collagen fibers and degrades slowly, allowing for in vivo tissue repair. A challenge with using a silk-based electrospun scaffold is achieving a homogenous distribution of cells throughout the scaffold. Importantly, tissue-engineered ligaments are deficient in type I collagen, a matrix protein critical for ligament functional properties. A macromolecular crowding (MMC) approach, where soluble macromolecules are added to culture medium, has been previously used to increase collagen deposition by fibroblasts and tenocytes in 2D cultures, but has not been used with electrospun scaffolds. This study i) investigated the use of a centrifugation-based dynamic seeding method to enhance cellular distribution within silk fibroin electrospun scaffolds and ii) evaluated the use of dextran sulfate (DxS) and Ficoll as MMC reagents to support collagen deposition by human dermal fibroblasts in actively seeded silk scaffolds. Assays for cellular metabolism and DNA content at 3 and 7 days post-seeding together demonstrated that cell numbers were increased in scaffolds subjected to short-term centrifugation (300g x 5 min) compared to passively seeded scaffolds. Analysis of cellular infiltration showed that exposure to centrifugation during seeding also led to a more uniform cellular distribution within scaffolds. For the first set of MMC experiments, medium was supplemented with DxS beginning on day 0 or 7. Collagen content normalized to DNA content showed a 51% increase relative to the control group with continuous DxS treatment and a 72% increase with the 7-day delay DxS for day 14 constructs. However, DNA content was significantly decreased by exposure to DxS compared to the control group (26% with continuous DxS or 19% with the 7-day delay). Selecting the 7-day delay MMC protocol for additional evaluation, the use of DxS or Ficoll as an MMC reagent was compared. Collagen content showed a 50% increase relative to the control group for DxS and a 54% increase for Ficoll. The increase in collagen content with Ficoll addition was supported by a 40% increase in DNA content. Overall, this study demonstrated that collagen accumulation could be enhanced in silk electrospun scaffolds actively seeded with fibroblasts by using DxS as an MMC reagent.

Bioengineering, University of California, San Diego (1); Biomedical Engineering, University of Minnesota (2); Orthopedic Surgery, University of California, San Diego (3); Radiology, University of California, San Diego (4); Sanford Consortium for Regenerative Medicine (5)

Decellularized extracellular matrix (ECM) hydrogels present a novel, clinical intervention for a myriad of regenerative medicine applications. In this paradigm, the source of ECM is typically the same tissue to which the treatment is applied; however, the need for tissue specific ECM sources has not been rigorously studied. We hypothesized, in a muscle regeneration model, that tissue specific ECM improves regeneration and function through preferentially stimulating physiologically relevant processes (e.g. progenitor cell proliferation and differentiation). One of three decellularized hydrogels: tissue specific skeletal muscle, non-specific cardiac muscle, and non mesoderm-derived lung, or saline were injected intramuscularly two days after notexin injection in male adult C57BL/6 mice (n=7 per time point) and muscle was harvested at days 5 and 14 for histological and gene expression analysis. All injectable hydrogels were decellularized using detergent methods and were controlled for donor characteristics (i.e. species, age). At day 5, the skeletal muscle ECM hydrogel significantly increased the density of Pax7+ and myogenin+ satellite cells in the muscle. Gene expression analysis at day 5 showed that both cardiac and skeletal muscle ECM hydrogels increased expression of genes implicated in muscle hypertrophy/growth. By day 14, both skeletal muscle and cardiac muscle ECM hydrogels improved muscle regeneration over saline and lung ECM hydrogels as shown through a shift in fiber cross sectional area distribution towards larger fibers (average fiber areas – saline: 564 ± 64 µm2; lung ECM: 545 ± 65 µm2; cardiac ECM: 623 ± 80 µm2; skeletal muscle ECM: 695 ± 94 µm2; data are mean ± SEM). This data indicates a potential role for muscle-specific regenerative capacity of decellularized, injectable muscle hydrogels. Further transcriptomic analysis of whole muscle mRNA is ongoing to understand mechanisms of tissue specificity in decellularized ECM hydrogels. This will lead to a greater understanding of the need (or lack thereof) for tissue specificity in regenerative medicine applications and elucidate potential ECM-mediated tissue repair mechanisms in vivo.

Macrophage activity is a central component of wound healing and tissue repair. In regenerating tissues, macrophage polarization progresses through a sequence of pro- and anti-inflammatory polarization states (e.g. the spectra of M1 and M2), which drives first pathogen clearance then tissue regeneration and remodeling. Though this process is tightly controlled in healthy tissues, this regulation is lost in many inflammatory disorders, e.g. type II diabetes. It is thus clear that control of macrophage activity is essential to promote successful healing in current regenerative medicine strategies, including immunomodulation via mesenchymal stem cell (MSC) therapies. Despite the importance of macrophage activity, the kinetics of macrophage polarization remain poorly characterized. Further, the mechanisms that control these dynamics during tissue repair have been described primarily qualitatively to date. To address this knowledge gap, we have created a fluorescent iNOS reporter RAW264.7 macrophage cell line that provides a real-time readout of polarization in response to external stimuli. Using this cell line, we found that macrophages treated with LPS produced a mean iNOS response that rose to a peak at 24 hours then exponentially decayed. However, our iNOS reporter has revealed that single cells exhibit a more switch-like response, in which polarizing cells rapidly begin expressing iNOS and maintain high expression levels. Rather than each cell gradually increasing iNOS levels, the number of macrophages expressing iNOS increased with time. To interrogate mechanisms responsible for macrophage polarization, we sought to quantify MAPK signaling within conditioned macrophages. Our population-averaged data, obtained via multiplexed immunoassay, show that LPS stimulation caused rapid and sustained phosphorylation of canonical MAPK signals, including Erk, p38, cJun, and Mek. However, ICC data suggest that only a fraction of total cells respond, mirroring our iNOS reporter data. In total, our analyses suggest that macrophage polarization is driven by the activity of a fraction of total cells. Our single cell macrophage polarization platform provides a novel technique for understanding how different therapeutic strategies modulates macrophage polarization in damaged tissue microenvironments. Moreover, its application to MSCs will lead to a new understanding of the mechanisms behind MSC therapeutic efficacy and new strategies for predicting MSC efficacy.

Stroke is the leading cause of disability and the fifth leading cause of death among American adults. With no Food and Drug Administration (FDA)-approved, restorative treatment options currently available to stroke patients, the regenerative capacity of a new class of autologous neural stem cells (NSCs) is enticing. Induced pluripotent stem cell (iPSC) derived NSCs (iNSCs) have been shown to promote neurocognitive and sensorimotor recovery in rodent stroke models by not only promoting endogenous cell survival through the secretion of regenerative trophic factors, but also by replacing lost or damaged cells through tissue integration. Despite these benefits, the overall survivability of iNSCs post-stroke is limited due to secondary injury cascades and resulting cytotoxic conditions. In this study, the efficacy of novel drug-loaded nanoparticles (NPs) in combination with iNSCs were tested in a translational pig ischemic stroke model. We hypothesized administration of drug-loaded, NPs would ameliorate acute cytotoxicity and promote enhanced survivability and long-term integration of iNSCs post-stroke. Ischemic stroke will be induced in 18 adult castrated male Landrace pigs by permanent middle cerebral artery occlusion (MCAO). Peripheral venous blood will be collected pre-stroke, 4, 12, and 24 hours post-stroke as well as 3, 5, 8, 12, 19, 35, 65, and 95 days post-stroke to evaluate subsequent changes in inflammatory markers IL-6, TNF-α, IFN-ϒ, SOD, and TBARS. We expect NP administration in combination with iNSC transplantation will lead to significant improvements at both acute and chronic time points post-stroke. By effectively alleviating the pro-inflammatory response and regenerating lost tissues, NP and iNSC combination therapies possess the potential to address the shortcomings of current stroke treatments.

Biomedical Engineering, University of Wisconsin-Madison (1); Orthopedics and Rehabilitation, University of Wisconsin-Madison (2); Materials Science, University of Wisconsin-Madison (3); Stem Cell and Regenerative Medicine Center, University of Wisconsin-Madison (4)

Human pluripotent stem cells (hPSCs) hold vast potential in regenerative medicine, as they can self-renew indefinitely and differentiate into clinically relevant cell types. However, current methods to expand and differentiate hPSCs at the scale required for cell therapies demand high doses of growth factors that are costly, have limited stability, and must be replenished frequently in culture. For example, standard media for expansion of undifferentiated hPSCs (e.g., Essential 8) contain basic fibroblast growth factor (bFGF) at 100 ng/mL concentrations. bFGF has a short half-life (~7 hrs at 37°C), necessitating that media be changed daily to maintain pluripotency. To address limitations of low bioactivity in protein delivery strategies, our group developed nanostructured mineral coatings that can bind, stabilize, and deliver a variety of therapeutic proteins. Here we hypothesized that stabilization and sustained release of bFGF from mineral-coated microparticles (MCMs) could maintain undifferentiated hPSCs while reducing bFGF usage in long-term culture.

MCMs were prepared by incubating hydroxyapatite powder in modified simulated body fluid for 5 days, lyophilized, and loaded by incubating in 1 μg/mL bFGF for 1 hr. To explore whether MCMs could prolong bFGF bioactivity in culture, we assessed pluripotency marker expression over time in hPSCs cultured in i) Essential 8 (E8), ii) E7 (i.e., E8 minus bFGF), and iii) E7+bFGF-loaded MCMs. We tested two methods of culture with bFGF-loaded MCMs: i) Indirect culture (MCMs releasing bFGF from Transwells) and ii) Direct culture (MCMs added directly to cells). hPSCs in E7 alone spontaneously differentiated by day 8, as shown by loss of colony morphology and reduced Oct4/Nanog expression. In contrast, hPSCs cultured with E7+bFGF-loaded MCMs had normal morphology and were >90% Oct4+/Nanog+ after 12 days in direct or indirect culture, comparable to hPSCs in E8 alone. We used Design of Experiments to optimize bFGF binding, to achieve >95% Oct4+/Nanog+ hPSCs at day 12 while minimizing bFGF usage. Compared to standard culture in E8, optimized MCMs did not require daily bFGF supplementation and thus reduced bFGF usage by 23.5% (indirect culture) and 81.3% (direct culture) while maintaining >95% Oct4+/Nanog+ hPSCs over >24 days. We posit that this strategy for protein stabilization and delivery may overcome limitations in biomanufacturing by reducing the need for costly growth factors in hPSC expansion and differentiation.

Biodegradable biomaterials exhibiting angiogenic properties are expected to improve vascularization for applications in tissue engineering and wound healing. Numerous approaches have been investigated, including using these materials as delivery vehicles for endogenous or exogenous growth factors and for engineering blood vessel constructs, however, achieving functional vascularization remains a challenge. The embryonic chorion represents a highly vascularized membrane in mammals, birds and reptiles, and clinically approved human-sourced amnion/chorion allografts have been shown to be pro-angiogenic. As an alternative to human-sourced chorion, we have previously identified FGF2-mediated pro-angiogenic properties of porcine aortic adventitia (pAdv) extracellular matrix (ECM). As yet another alternative, here we hypothesized that decellularized chick chorioallantoic membrane (dCAM) retains bioactive signals capable of regenerating small and large diameter capillaries. Decellularized bioscaffolds made from CAM had minimal DNA compared to respective native tissues, validating the decellularization process. Pepsin-digested lyophilized dCAM powder formed hydrogels within 90 min at physiological pH, temperature, rate similar to other ECM biomaterials. dCAM bioscaffolds increased proliferation of human and murine endothelial cells, directed endothelial cell migration, and promoted formation of tube-like structures in vitro, and they increased vascular invasion and capillary formation in the chick CAM assay and mouse subcutaneous plug assay in vivo. This study demonstrated that dCAM hydrogels retain pro-angiogenic signals and supports the feasibility of using dCAM bioscaffolds for both biological discovery and potential translation towards vascular regeneration in clinical applications.